Alpha-amylase

ABSTRACT

This invention relates to a novel alpha-amylase, a process for its preparation and the use of the amylase. 
     The invention relates to a newly identified polynucleotide sequence from  Alicyclobacillus pohliae  comprising a gene that encodes the novel alpha-amylase enzyme. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional protein of the gene. The invention also relates to methods of using these proteins in industrial processes, for example in food industry, such as the baking industry. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins and cells.

FIELD OF INVENTION

This invention involves a novel alpha-amylase, a process for itspreparation and the use of the amylase.

The invention relates to a newly identified polynucleotide sequencecomprising a gene that encodes the novel alpha-amylase enzyme. Theinvention features the full length coding sequence of the novel gene aswell as the amino acid sequence of the full-length functional protein ofthe gene. The invention also relates to methods of using these proteinsin industrial processes, for example in food industry, such as thebaking industry. Also included in the invention are cells transformedwith a polynucleotide according to the invention suitable for producingthese proteins and cells. The invention relates to a method ofmanufacturing the polynucleotide according to the invention. Theinvention further relates to a method for manufacturing the polypeptideaccording to the invention.

BACKGROUND OF THE INVENTION

Studies on bread staling have indicated that the starch fraction inbread recrystallizes during storage, thus causing an increase in crumbfirmness, which may be measured as an increase in hardness of breadslices.

The present invention relates to an alpha-amylase. Alpha-amylases havebeen used in industry for a long time.

Alpha-amylases have traditionally been provided through the inclusion ofmalted wheat or barley flour and give several advantages to the baker.Alpha-amylase is used to give satisfactory gas production and gasretention during dough leavening and to give satisfactory crust color.This means that if this enzyme is not used in sufficient amount, thevolume, texture, and appearance of the loaf are substantially impaired.Alpha-amylase occurs naturally within the wheat crop itself, measuredroutinely by Hagberg Falling Number (ICC method 107), and steps aretaken to minimise such variations by the addition of alpha-amylase atthe mill and through the use of specialty ingredients at the bakery asthe enzyme is of such critical importance.

In more recent times, alpha-amylase from cereal has been largelyreplaced with enzymes from microbial sources, including fungal andbacterial sources. Through use of biotechnology in strain selection,fermentation and processing, enzymes can be prepared from such microbialsources and this brings advantage over malt flour because the enzyme isof more controlled quality, relatively pure and more cost effective inuse.

The properties of alpha-amylases, and their technological effects, dohowever show important differences. Besides giving influence to gasproduction, gas retention and crust color, alpha-amylase can havebearing on the shelf-life of the baked product.

Starch within the wheat flour contains two principal fractions, amyloseand amylopectin, and these are organised in the form of starch granules.A proportion of these granules from hard-milling wheat varieties become“damaged”, with granules splitting apart as a consequence of the energyof milling. In the process of baking, the starch granules gelatinise;this process involves a swelling of the granule by the uptake of waterand a loss of the crystalline nature of the granule; in particularamylopectins within the native granule are known to exist ascrystallites and these molecules dissociate and lose crystallinityduring gelatinisation. Once the bread has been baked, amylopectinrecrystallises slowly over a numbers of days and it is thisrecrystallisation, or retrogradation of starch, that is regarded asbeing the principal cause of bread staling.

These varying forms of the starch and their interaction withalpha-amylase dictate the role the enzyme has with respect to bakingtechnology. Alpha-amylase from fungal sources, most typically comingfrom Aspergillus species, acts principally on damaged starch during themixing of dough and throughout fermentation/proof. The low heatstability of the enzyme means that the enzyme is inactivated duringbaking and, critically before starch gelatinisation has taken place,such that there is little or no breakdown of the starch from theundamaged fraction. As a consequence, fungal amylase is useful inproviding sugars for fermentation and color, but has practically novalue in extending shelf-life. Bacterial alpha-amylase, most typicallyfrom Bacillus amyloliquifaciens, on the other hand does bring extendedtemperature stability and activity during the baking of bread and whilestarch is undergoing gelatinisation. Bacterial amylase then leads tomore extensive modification of the starch and, in turn, the qualities ofthe baked bread; in particular the crumb of the baked bread can beperceptibly softer throughout shelf-life and can permit the shelf-lifeto be increased. However, while bacterial alpha-amylase can be usefulwith regard to shelf-life extension, it is difficult to use practicallyas small over-doses lead to an unacceptable crumb structure of large andopen pores, while the texture can become too soft and “gummy”.

The inventor has identified an alpha amylase from a particular bacterialsource that has a thermostability falling inbetween typical fungal andbacterial alpha amylases. The thermostability of this enzyme is higherthan fungal alpha amylase, thereby allowing greater activity onamylopectin during and after gelatinisation, but it is not acting aslong into the baking process as the typical bacterial amylases and isnot over digesting the starch.

U.S. Pat. No. 4,598,048 describes the preparation of a maltogenicamylase enzyme. U.S. Pat. No. 4,604,355 describes a maltogenic amylaseenzyme, preparation and use thereof. U.S. Pat. No. RE38,507 describes anantistaling process and agent. The product described in U.S. Pat. No.RE38,507 is used in industry under the trade name Novamyl®.

It was set out to find an organism able to produce an improvedalpha-amylase. As a result the inventor has identified theAlicyclobacillus pohliae NCIMB14276 strain which was discovered in theAntarctic. This is a new strain, it was not previously used as a sourcefor an alpha-amylase which has improved properties.

SUMMARY OF THE INVENTION

The present invention relates to polypeptides. The invention provides anovel alpha-amylase that may be used for retarding staling of bakedproducts such as bread and cake. The invention further provides novelpolynucleotides encoding the novel alpha-amylase enzyme.

Accordingly, the invention relates to:

-   a polynucleotide encoding for a polypeptide having alpha-amylase    activity comprising:    -   (a) a polynucleotide sequence encoding a polypeptide having an        amino acid sequence as set out in SEQ ID NO: 2 or having an        amino acid sequence as set out in amino acids 34 to 719 of SEQ        ID NO: 2; or    -   (b) a polynucleotide sequence encoding a polypeptide having at        least 99.5% identity to a polypeptide having an amino acid        sequence as set out in amino acids 34 to 719 of SEQ ID NO: 2; or    -   (c) a polynucleotide sequence as set out in nucleotides 100 to        2157 of SEQ ID NO: 1 or SEQ ID NO: 3; or    -   (d) a polynucleotide sequence as set out in SEQ ID NO: 1 or SEQ        ID NO: 3.

Further the invention concerns:

-   an alpha-amylase polypeptide comprising:    -   (a) an amino acid sequence as set out in amino acids 34 to 719        of SEQ ID NO: 2; or    -   (b) an amino acid sequence having at least 99.5% identity to an        amino acid sequence as set out in amino acids 34 to 719 of SEQ        ID NO: 2; or    -   (c) an amino acid sequence encoded by a polynucleotide as set        out in nucleotides 100 to 2157 of SEQ ID NO: 1 or SEQ ID NO: 3;        or    -   (d) the amino acid sequence according to (c), wherein the        polynucleotide is produced by Alicyclobacillus pohliae        NCIMB14276; or    -   (e) an amino acid sequence having at least 70% identity to an        amino acid sequence as set out in amino acids 34 to 719 of SEQ        ID NO: 2 and having at least one of Asp at position 184, Ala at        position 297, Thr at position 368 and Asn at position 489, said        positions being defined with reference to SEQ ID NO: 2; or    -   (f) an amino acid sequence having at least 70% identity to an        amino acid sequence as set out in amino acids 34 to 719 of SEQ        ID NO: 2 and having at least one of Asp at position 184, Ala at        position 297, Thr at position 368 and Asn at position 489, said        positions being defined with reference to SEQ ID NO: 2 and said        amino acid sequence characterized in that when used to prepare a        baked product having a least 5 wt % sugar based on flour, said        baked product has reduced hardness after storage in comparison        with a baked product prepared without use of said amino acid        sequence.

In another aspect the invention relates to a vector comprising thepolynucleotide sequence according to the invention. The invention alsorelates to a recombinant host cell comprising the polynucleotideaccording to the invention. The invention relates to a method ofmanufacturing the polynucleotide according to the invention. Theinvention further relates to a method for manufacturing the polypeptideaccording to the invention. The invention relates to the use of saidpolypeptide in food manufacturing. The invention also relates to anenzyme composition. The invention also relates to a method to prepare adough and to a dough comprising the polypeptide according to theinvention or the enzyme composition according to the invention.

The invention also relates to a method to prepare a baked productcomprising the step of baking the dough according to the invention.

The invention further relates to a baked product.

The invention further relates to a method to produce a polypeptidecomprising the use of Alicyclobacillus pohliae NCIMB14276.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 sets out the plasmid map of pGBB09.

FIG. 2 sets out the plasmid map of pGBB09DSM-AM1.

FIG. 3 Sets out SEQ ID NO: 1.

FIG. 4 Sets out SEQ ID NO: 2.

FIG. 5 Sets out SEQ ID NO: 3.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 sets out the polynucleotide sequence from Alicyclobacilluspohliae NCIMB14276 encoding the wild type signal sequence (set out innucleotides 1 to 99), the alpha-amylase according to the invention (setout in nucleotides 100 to 2157), and a stop codon at the 3′-terminus(set out in nucleotides 2157 to 2160).

SEQ ID NO: 2 sets out the amino acid sequence of the Alicyclobacilluspohliae NCIMB14276 wild type signal sequence (set out in amino acids 1to 33) and the alpha-amylase according to the invention (set out inamino acids 34 to 719). Also referred to herein as DSM-AM protein.

SEQ ID NO: 3 sets out a codon optimised polynucleotide sequence fromAlicyclobacillus pohliae NCIMB14276 encoding the wild type signalsequence (set out in nucleotides 1 to 99), the alpha-amylase accordingto the invention (set out in nucleotides 100 to 2157), and a stop codonat the 3′-terminus (set out in nucleotides 2157 to 2160).

DETAILED DESCRIPTION OF THE INVENTION

Throughout the present specification and the accompanying claims thewords “comprise” and “include” and variations such as “comprises”,“comprising”, “includes” and “including” are to be interpreted as openand inclusive. That is, these words are intended to convey the possibleinclusion of other elements or integers not specifically recited, wherethe context allows.

Throughout the present specification and the accompanying claims thewording “nucleotides 100 to 2157” means nucleotides 100 up to andincluding 2157. Throughout the present specification and theaccompanying claims the wording “amino acids 34 to 719” means aminoacids 34 up to and including 719.

The terms “polypeptide having an amino acid sequence as set out in aminoacids 34 to 719 of SEQ ID NO: 2, “the mature polypeptide as set out inSEQ ID NO: 2” and “mature DSM-AM” are used interchangeably herein.

The terms “polypeptide having at least 99.5% identity to a polypeptidehaving an amino acid sequence as set out in amino acids 34 to 719 of SEQID NO: 2”, “mature polypeptide according to the invention”, “matureenzyme according to the invention”, “amylolytic enzyme according to theinvention”, “alpha-amylase enzyme according to the invention”,“alpha-amylase according to the invention” and “polypeptide according tothe invention” are used interchangeably herein.

The terms “polypeptide having at least 70% identity to a polypeptidehaving an amino acid sequence as set out in amino acids 34 to 719 of SEQID NO: 2”, “mature polypeptide according to the invention”, “matureenzyme according to the invention”, “amylolytic enzyme according to theinvention”, “alpha-amylase enzyme according to the invention”,“alpha-amylase according to the invention” and “polypeptide according tothe invention” are used interchangeably herein.

The terms “according to the invention” and “of the invention” are usedinterchangeably herein.

The terms “DSM-AM gene”, “alpha-amylase gene according to theinvention”, “AM gene” and “polynucleotide according to SEQ ID NO: 1” areused interchangeably herein.

The term “polynucleotide according to the invention” includes SEQ ID NO:1 and SEQ ID NO: 3.

In the context of the present invention “mature polypeptide” is definedherein as a polypeptide having alpha-amylase activity that is in itsfinal form following translation and any post-translationalmodifications, including N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. The process of maturation maydepend on the particular expression vector used, the expression host andthe production process.

To confirm the polynucleotide sequence of the DSM-AM gene from theAlicyclobacillus pohliae NCIMB14276, the whole genome of A. pohliaeNCIMB14276 was sequenced. The results hereof confirmed thepolynucleotide, encoding the DSM-AM protein, is as disclosed in SEQ IDNO: 1. From this the 719 amino acid sequence of the DSM-AM protein asset out in SEQ ID NO: 2 was confirmed. The first 33 amino acids,starting from the N′-terminus of the DSM-AM protein, belong to thesignal sequence.

Polynucleotides

The invention relates to a polynucleotide encoding for a polypeptidehaving alpha-amylase activity comprising:

-   -   (a) a polynucleotide sequence encoding a polypeptide having an        amino acid sequence as set out in amino acids 34 to 719 of SEQ        ID NO: 2; or    -   (b) a polynucleotide sequence encoding a polypeptide having at        least 99.5% identity to a polypeptide having an amino acid        sequence as set out in amino acids 34 to 719 of SEQ ID NO: 2; or    -   (c) a polynucleotide sequence as set out in nucleotides 100 to        2157 of SEQ ID NO: 1 or SEQ ID NO: 3; or    -   (d) a polynucleotide sequence as set out in SEQ ID NO: 1 or SEQ        ID NO: 3.

In an aspect, a polynucleotide of the invention is an isolatedpolynucleotide comprising:

-   -   (a) a polynucleotide sequence as set out in nucleotides 100 to        2157 of the polynucleotide sequence of SEQ ID NO: 1 or 3        (inclusive of nucleotides 100 and 2157, for the avoidance of        doubt); or    -   (b) a polynucleotide sequence encoding a polypeptide having an        amino acid sequence as set out in SEQ ID NO: 2 or having an        amino acid sequence as set out in amino acids 34 to 719 of SEQ        ID NO: 2 (inclusive of amino acids 34 and 719, for the avoidance        of doubt); or    -   (c) a polynucleotide sequence as set out in SEQ ID NO:1 or SEQ        ID NO:3.

In one aspect such isolated polynucleotide can be obtainedsynthetically, e.g. by solid phase synthesis or by other methods knownto the person skilled in the art.

The sequences according to SEQ ID NO: 1 and SEQ ID NO: 3 include thenucleotides encoding the mature polypeptide according to the inventionand the wild type signal sequence. SEQ ID NO: 2 includes the maturepolypeptide according to the invention and the wild type signalsequence.

An “isolated polynucleotide” or “isolated nucleic acid” is a DNA or RNAthat is not immediately contiguous with both of the coding sequenceswith which it is immediately contiguous (one on the 5′ end and one onthe 3′ end) in the naturally occurring genome of the organism from whichit is obtained. Thus, in one embodiment, an isolated polynucleotideincludes some or all of the 5′ non-coding (e.g., promotor) sequencesthat are immediately contiguous to the coding sequence. The termtherefore includes, for example, a recombinant DNA that is incorporatedinto a vector, into an autonomously replicating plasmid or virus, orinto the genomic DNA of a prokaryote or eukaryote, or which exists as aseparate molecule (e.g., a cDNA or a genomic DNA fragment produced byPCR or restriction endonuclease treatment) independent of othersequences. It also includes a recombinant DNA that is part of a hybridgene encoding an additional polypeptide that is substantially free ofcellular material, viral material, or culture medium (when produced byrecombinant DNA techniques), or chemical precursors or other chemicals(when chemically synthesized). Moreover, an “isolated polynucleotidefragment” is a polynucleotide fragment that is not naturally occurringas a fragment and would not be found in the natural state.

Polynucleotides of the invention also include polynucleotides whichcomprise certain variant sequences of the coding sequence of SEQ ID NO:1 or 3 and which can encode a functional alpha-amylase. Such variantsequences thus encode polypeptides with alpha-amylase activity.

A polynucleotide sequence of the invention will generally comprisesequence encoding a polypeptide having at least about 99.5% sequenceidentity to a polypeptide having an amino acid sequence as set out inamino acids 34 to 719 of SEQ ID NO: 2 as calculated over the fulllengths of those sequences.

The coding sequence of SEQ ID NO: 1 or 3 may be modified by nucleotidesubstitutions. The polynucleotide of SEQ ID NO: 1 or 3 may alternativelyor additionally be modified by one or more insertions and/or deletionsand/or by an extension at either or both ends. The modifiedpolynucleotide encodes a polypeptide which has alpha-amylase activity.

In an embodiment of the polynucleotide according to the invention thepolynucleotide is produced by Alicyclobacillus pohliae NCIMB14276. Asused herein, the terms “polynucleotide” or “nucleic acid molecule” andthe like are intended to include DNA molecules (e.g., cDNA or genomicDNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNAgenerated using nucleotide analogs. The polynucleotide molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA. The polynucleotide may be synthesized so that it includes syntheticor modified nucleotides. A number of different types of modification topolynucleotides are known in the art. These include methylphosphonateand phosphorothioate backbones, addition of acridine or polylysinechains at the 3′ and/or 5′ ends of the molecule. Such oligonucleotidescan be used, for example, to prepare polynucleotides that have alteredbase-pairing abilities or increased resistance to nucleases.

For the purposes of the present invention, it is to be understood thatthe polynucleotides described herein may be modified by any methodavailable in the art. Such modifications may be carried out, for exampleto increase the extent to which a polynucleotide of the invention may beexpressed in a suitable host cell.

A polynucleotide of the invention, such as a DNA polynucleotide, may beproduced recombinantly, synthetically, or by any means available tothose of skill in the art. They may also be cloned by standardtechniques. A polynucleotide of the invention is typically provided inisolated and/or purified form.

Polynucleotides may be produced using recombinant means, for exampleusing PCR (polymerase chain reaction) cloning techniques. This willinvolve making a pair of primers (e.g. of about 15-30 nucleotides) to aregion of the polynucleotide which it is desired to amplify, bringingthe primers into contact with mRNA or cDNA obtained from a suitablecell, performing a polymerase chain reaction under conditions whichbring about amplification of the desired region, isolating andrecovering the amplified DNA. The primers may be designed to containsuitable restriction enzyme recognition sites so that the amplified DNAcan conveniently be cloned into a suitable cloning vector.

Such techniques may be used to obtain all or part of the polynucleotideof SEQ ID NO: 1 or 3 described herein or variants thereof.

Polynucleotides which do not have 100% sequence identity to the sequenceof SEQ ID NO: 1 or 3 but which nevertheless fall within the scope of theinvention may be obtained in a number of ways. For example,polynucleotides may be obtained by an appropriate mutagenesis technique,such as site-directed mutagenesis of SEQ ID NO: 1 or 3. This may beuseful where, for example, silent codon changes are required tosequences to optimize codon preferences for a particular host cell inwhich the polynucleotide sequences are being expressed. Other sequencechanges may be desired in order to introduce restriction enzymerecognition sites, or to alter the property or function of thepolypeptides encoded by the polynucleotides.

To increase the likelihood that the introduced enzyme is expressed inactive form in a cell of the invention, the corresponding encodingnucleotide sequence may be adapted to optimise its codon usage to thatof the chosen host cell, for example SEQ ID NO: 3. Several methods forcodon optimisation are known in the art. A preferred method to optimisecodon usage of the nucleotide sequences to that of the chosen host cellis a codon pair optimization technology as disclosed in WO2006/077258and/or WO2008/000632. WO2008/000632 addresses codon-pair optimization.Codon-pair optimisation is a method wherein the nucleotide sequencesencoding a polypeptide are modified with respect to their codon-usage,in particular the codon-pairs that are used, to obtain improvedexpression of the nucleotide sequence encoding the polypeptide and/orimproved production of the encoded polypeptide. Codon pairs are definedas a set of two subsequent triplets (codons) in a coding sequence.

As a simple measure for gene expression and translation efficiency,herein, the Codon Adaptation Index (CAI), as described in Xuhua Xia,Evolutionary Bioinformatics 2007, 3: 53-58, is used. The index uses areference set of highly expressed genes from a species to assess therelative merits of each codon, and a score for a gene is calculated fromthe frequency of use of all codons in that gene. The index assesses theextent to which selection has been effective in moulding the pattern ofcodon usage. In that respect it is useful for predicting the level ofexpression of a gene, for assessing the adaptation of viral genes totheir hosts, and for making comparisons of codon usage in differentorganisms. The index may also give an approximate indication of thelikely success of heterologous gene expression. In the codon pairoptimized genes according to the invention, the CAI is 0.6 or more, 0.7or more, 0.8 or more, 0.85 or more, 0.87 or more 0.90 or more, 0.95 ormore, or about 1.0.

The invention further provides double stranded polynucleotidescomprising a polynucleotide of the invention and its complement.

Polynucleotides, probes or primers of the invention may carry arevealing label. Suitable labels include radioisotopes such as ³²P or³⁵S, enzyme labels, or other protein labels such as biotin. Such labelsmay be added to polynucleotides, probes or primers of the invention andmay be detected using techniques known per se.

Polypeptides

The invention provides an (isolated) polypeptide having starch degradingactivity. The invention further relates to a method for manufacturingthe polypeptide according to the invention.

The terms “peptide” and “oligopeptide” are considered synonymous (as iscommonly recognized) and each term can be used interchangeably as thecontext requires to indicate a chain of at least two amino acids coupledby peptidyl linkages. The word “polypeptide” (or protein) is used hereinfor chains containing more than seven amino acid residues. Alloligopeptide and polypeptide formulas or sequences herein are writtenfrom left to right and in the direction from amino terminus to carboxyterminus. The three-letter code of amino acids used herein is commonlyknown in the art and can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual, 3^(rd) ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 2001).

The invention relates to an alpha-amylase polypeptide comprising:

-   -   (a) an amino acid sequence as set out in amino acids 34 to 719        of SEQ ID NO: 2; or    -   (b) an amino acid sequence having at least 99.5% identity to an        amino acid sequence as set out in amino acids 34 to 719 of SEQ        ID NO: 2; or    -   (c) an amino acid sequence encoded by a polynucleotide as set        out in nucleotides 100 to 2157 of SEQ ID NO: 1 or SEQ ID NO: 3;        or    -   (d) an amino acid sequence having at least 70% identity to an        amino acid sequence as set out in amino acids 34 to 719 of SEQ        ID NO: 2 and having at least one of Asp at position 184, Ala at        position 297, Thr at position 368 and Asn at position 489, said        positions being defined with reference to SEQ ID NO: 2; or    -   (e) an amino acid sequence having at least 70% identity to an        amino acid sequence as set out in amino acids 34 to 719 of SEQ        ID NO: 2 and having at least one of Asp at position 184, Ala at        position 297, Thr at position 368 and Asn at position 489, said        positions being defined with reference to SEQ ID NO: 2 and said        amino acid sequence characterized in that when used to prepare a        baked product having a least 5 wt % sugar based on flour, said        baked product has reduced hardness after storage in comparison        with a baked product prepared without use of said amino acid        sequence.

In an embodiment, the invention relates to an isolated polypeptidecomprising:

-   -   (a) amino acid sequence as set out in amino acids 34 to 719 of        SEQ ID NO: 2; or or    -   (b) an amino acid sequence encoded by a polynucleotide as set        out in nucleotides 100 to 2157 of SEQ ID NO: 1 or 3; or    -   (c) the amino acid sequence according to (b), wherein the        polynucleotide is produced by Alicyclobacillus pohliae        NCIMB14276; or    -   (d) an amino acid sequence encoded by a polynucleotide sequence        as set out in SEQ ID NO: 1 or SEQ ID NO: 3.

The polypeptide of the invention comprises the amino acid sequencehaving at least 99.5% identity, preferably at least 99.6% identity,preferably at least 99.7% identity preferably at least 99.8% identity,preferably at least 99.9% identity to a polypeptide having an amino acidsequence as set out in amino acids 34 to 719 of SEQ ID NO: 2 which hasalpha-amylase activity. In general, the naturally occurring amino acidsequence shown in amino acids 34 to 719 of SEQ ID NO: 2 is preferred.

In an aspect the polypeptide of the invention comprises an amino acidsequence having at least 70% identity, in an aspect at least 80%identity, in an aspect at least 85% identity, in an aspect at least 90%identity, in an aspect at least 95% identity to an amino acid sequenceas set out in amino acids 34 to 719 of SEQ ID NO: 2 and having at leastone of Asp at position 184, Ala at position 297, Thr at position 368 andAsn at position 489, said positions being defined with reference to SEQID NO: 2.

As is known to the person skilled in the art it is possible that the N-and/or C-termini of SEQ ID NO: 2 or of the mature polypeptide in theamino acid sequence according to SEQ ID NO: 2 (as set out in amino acids34 to 719) might be heterogeneous, due to variations in processingduring maturation. In particular such processing variations might occurupon overexpression of the polypeptide. In addition, exo-proteaseactivity might give rise to heterogeneity. The extent to whichheterogeneity occurs depends also on the host and fermentation protocolsthat are used. Such C-terminal processing artefacts might lead toshorter polypeptides or longer polypeptides as indicated with SEQ ID NO:2 or with the mature polypeptide in the amino acid sequence according toSEQ ID NO: 2. As a result of such processing variations the N-terminusmight also be heterogeneous. Processing variants at the N-terminus couldbe due to alternative cleavage of the signal sequence by signalpeptidases.

In a further aspect, the invention provides an isolated polynucleotideencoding the polypeptide according to SEQ ID NO: 2 or of the maturepolypeptide in the amino acid sequence according to SEQ ID NO: 2 whichcontain additional residues and start at position −1, or −2, or −3 etc.Alternatively, it might lack certain residues and as a consequence startat position 2, or 3, or 4 etc. Also additional residues may be presentat the C-terminus, e.g. at position 720, 721 etc. Alternatively, theC-terminus might lack certain residues and as a consequence end atposition 718, or 717 etc.

The polypeptide of the invention preferably has at 99.5% sequenceidentity to the sequence set out in SEQ ID NO: 2.

The sequence of the polypeptide of SEQ ID NO: 2 can thus be modified toprovide polypeptides of the invention. Amino acid substitutions may bemade, for example, 1, 2, 3 or 4 substitutions. The modified polypeptideretains activity as an alpha amylase.

In an aspect the polypeptide of the invention has at least 70% identity,in an aspect at least 80% identity, in an aspect at least 85% identity,in an aspect at least 90% identity, in an aspect at least 95% identity,in an aspect at least 99.5% identity to a polypeptide having an aminoacid sequence as set out in SEQ ID NO: 2 or having an amino acidsequence as set out in amino acids 34 to 719 of SEQ ID NO: 2.

Preferably, such an polypeptide has an amino acid sequence which, whenaligned with the amino acid sequence as set in SEQ ID NO 2, comprises atleast one of Asp at position 184, Ala at position 297, Thr at position368 and Asn at position 489, said positions being defined with referenceto SEQ ID NO: 2. Preferably such an alpha-amylase comprises at least Alaat position 297 said position being defined with reference to SEQ ID NO:2.

In an aspect the polypeptide of the invention may comprise at least twoof Asp at position 184, Ala at position 297, Thr at position 368 and Asnat position 489, said positions being defined with reference to SEQ IDNO: 2. Preferably such a polypeptide comprises at least: Asp at position184 and Ala at position 297; at least Ala at position 297 and Thr atposition 368; or at least Ala at position 297 and Asn at position 489,all of said positions being defined with reference to SEQ ID NO: 2.

In an aspect the polypeptide of the invention may comprise at leastthree of Asp at position 184, Ala at position 297, Thr at position 368and Asn at position 489, said positions being defined with reference toSEQ ID NO: 2. Preferably, such a polypeptide comprises at least: Ala atposition 297, Thr at position 368 and Asn at position 489; Asp atposition 184, Ala at position 297 and Thr at position 368; or Asp atposition 184, Ala at position 297 and Asn at position 489, all of saidpositions being defined with reference to SEQ ID NO: 2.

In an aspect the polypeptide of the invention may comprise Asp atposition 184, Ala at position 297, Thr at position 368 and Asn atposition 489, all of said positions being defined with reference to SEQID NO: 2.

In an aspect the polypeptide of the invention comprises an amino acidsequence having at least 80% identity to an amino acid sequence as setout in amino acids 34 to 719 of SEQ ID NO: 2 and having at least one ofAsp at position 184, Ala at position 297, Thr at position 368 and Asn atposition 489, said positions being defined with reference to SEQ ID NO:2.

In an aspect the polypeptide of the invention comprises an amino acidsequence having at least 85% identity to an amino acid sequence as setout in amino acids 34 to 719 of SEQ ID NO: 2 and having at least one ofAsp at position 184, Ala at position 297, Thr at position 368 and Asn atposition 489, said positions being defined with reference to SEQ ID NO:2.

In an aspect the polypeptide of the invention comprises an amino acidsequence having at least 90% identity to an amino acid sequence as setout in amino acids 34 to 719 of SEQ ID NO: 2 and having at least one ofAsp at position 184, Ala at position 297, Thr at position 368 and Asn atposition 489, said positions being defined with reference to SEQ ID NO:2.

In an aspect the polypeptide of the invention comprises an amino acidsequence having at least 95% identity to an amino acid sequence as setout in amino acids 34 to 719 of SEQ ID NO: 2 and having at least one ofAsp at position 184, Ala at position 297, Thr at position 368 and Asn atposition 489, said positions being defined with reference to SEQ ID NO:2.

In an aspect the polypeptide of the invention comprises an amino acidsequence having at least 80% identity to an amino acid sequence as setout in amino acids 34 to 719 of SEQ ID NO: 2 and having Asp at position184, Ala at position 297, Thr at position 368 and Asn at position 489,said positions being defined with reference to SEQ ID NO: 2.

In an aspect the polypeptide of the invention comprises an amino acidsequence having at least 85% identity to an amino acid sequence as setout in amino acids 34 to 719 of SEQ ID NO: 2 and having Asp at position184, Ala at position 297, Thr at position 368 and Asn at position 489,said positions being defined with reference to SEQ ID NO: 2.

In an aspect the polypeptide of the invention comprises an amino acidsequence having at least 90% identity to an amino acid sequence as setout in amino acids 34 to 719 of SEQ ID NO: 2 and having Asp at position184, Ala at position 297, Thr at position 368 and Asn at position 489,said positions being defined with reference to SEQ ID NO: 2.

In an aspect the polypeptide of the invention comprises an amino acidsequence having at least 95% identity to an amino acid sequence as setout in amino acids 34 to 719 of SEQ ID NO: 2 and having Asp at position184, Ala at position 297, Thr at position 368 and Asn at position 489,said positions being defined with reference to SEQ ID NO: 2.

In an aspect the polypeptide of the invention comprises an amino acidsequence having at least 99.5% identity to an amino acid sequence as setout in amino acids 34 to 719 of SEQ ID NO: 2 and having Asp at position184, Ala at position 297, Thr at position 368 and Asn at position 489,said positions being defined with reference to SEQ ID NO: 2.

In an aspect the polypeptide of the invention comprises an amino acidsequence having at least 70% identity to an amino acid sequence as setout in amino acids 34 to 719 of SEQ ID NO: 2 and having at least one ofAsp at position 184, Ala at position 297, Thr at position 368 and Asn atposition 489, said positions being defined with reference to SEQ ID NO:2 and said amino acid sequence characterized in that when used toprepare a baked product having a least 5 wt % sugar based on flour, saidbaked product has reduced hardness after storage in comparison with abaked product prepared without use of said amino acid sequence.

The one or more amino acids of the polypeptide according to theinvention may be substituted in order to improve the expression in ahost cell. In addition one or more amino acids of the protein accordingto the invention may be substituted to change the enzymes specificactivity or thermal stability.

Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulphur-containing sidechains is cysteine and methionine.

Preferred conservative amino acids substitution groups include:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine. Substitutional variants of theamino acid sequence disclosed herein are those in which at least oneresidue in the disclosed sequences has been removed and a differentresidue inserted in its place. Preferably, the amino acid change isconservative. Preferred conservative substitutions for each of thenaturally occurring amino acids include: Ala to ser; Arg to lys; Asn togln or his; Asp to glu; Cys to ser or ala; Gln to asn; Glu to asp; Glyto pro; His to asn or gln; He to leu or val; Leu to ile or val; Lys toarg; gln or glu; Met to leu or ile; Phe to met, leu or tyr; Ser to thr;Thr to ser; Trp to tyr; Tyr to trp or phe; and, Val to ile or leu.

Polypeptides of the invention may be in a substantially isolated form.It will be understood that the polypeptide may be mixed with carriers ordiluents which will not interfere with the intended purpose of thepolypeptide and still be regarded as substantially isolated. Thepolypeptide of the invention may also be in a substantially purifiedform, in which case it will generally comprise the polypeptide in apreparation in which more than 50%. e.g. more than 80%, 90%, 95% or 99%,by weight of the polypeptide in the preparation is a polypeptide of theinvention.

For example, recombinantly produced polypeptides and proteins producedin host cells are considered isolated for the purpose of the inventionas are native or recombinant polypeptides which have been substantiallypurified by any suitable technique such as, for example, the single-steppurification method disclosed in Smith and Johnson, Gene 67:31-40(1988).

The polypeptide of the invention may be chemically modified, e.g.post-translationally modified. For example, they may be glycosylated orcomprise modified amino acid residues. They may also be modified by theaddition of Histidine residues or a T7 tag to assist their purificationor by the addition of a signal sequence to promote their secretion froma cell. Such modified polypeptides and proteins fall within the scope ofthe term “polypeptide” of the invention.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, an appropriate secretion signal sequence maybe fused to the polynucleotide of the invention. The signals may beendogenous to the polypeptide or they may be heterologous signals.

The polypeptide according to the invention may be produced in a modifiedform, such as a fusion protein, and may include not only secretionsignals but also additional heterologous functional regions. Thus, forinstance, a region of additional amino acids, particularly charged aminoacids, may be added to the N-terminus of the polypeptide to improvestability and persistence in the host cell, during purification orduring subsequent handling and storage. Also, peptide moieties may beadded to the polypeptide to facilitate purification.

Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

Sequence Identity

The terms “homology”, “percent identity”, “percent homology” and“percentage of identity” are used interchangeably herein. For thepurpose of this invention, it is defined here that in order to determinethe percent homology of two amino acid sequences or of twopolynucleotide sequences (also referred to herein as nucleic acidsequences), the sequences are aligned for optimal comparison purposes.In order to optimize the alignment between the two sequences gaps may beintroduced in any of the two sequences that are compared. Such alignmentcan be carried out over the full length of the sequences being compared.Alternatively, the alignment may be carried out over a shorter length,for example over about 20, about 50, about 100 or more nucleicacids/based or amino acids. The percent homology or percent identity isthe percentage of identical matches between the two sequences over thereported aligned region.

A comparison of sequences and determination of percent identity betweentwo sequences can be accomplished using a mathematical algorithm. Theskilled person will be aware of the fact that several different computerprograms are available to align two sequences and determine the homologybetween two sequences (Kruskal, J. B. (1983) An overview of sequencecomparison In D. Sankoff and J. B. Kruskal, (ed.), Time warps, stringedits and macromolecules: the theory and practice of sequencecomparison, pp. 1-44 Addison Wesley). The percent identity between twoamino acid sequences or between two nucleotide sequences may bedetermined using the Needleman and Wunsch algorithm for the alignment oftwo sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol.48, 443-453). Both amino acid sequences and polynucleotide sequences canbe aligned by the algorithm. The Needleman-Wunsch algorithm has beenimplemented in the computer program NEEDLE. For the purpose of thisinvention the NEEDLE program from the EMBOSS package was used (version2.8.0 or higher, EMBOSS: The European Molecular Biology Open SoftwareSuite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16,(6) pp 276-277, http://emboss.bioinformatics.nl/). For protein sequencesEBLOSUM62 is used for the substitution matrix. For nucleotide sequence,EDNAFULL is used. The optional parameters used are a gap-open penalty of10 and a gap extension penalty of 0.5. The skilled person willappreciate that all these different parameters will yield slightlydifferent results but that the overall percentage identity of twosequences is not significantly altered when using different algorithms.

After alignment by the program NEEDLE as described above the percentageof identity between a query sequence and a sequence of the invention iscalculated as follows: Number of corresponding positions in thealignment showing an identical aminoacid or identical nucleotide in bothsequences divided by the total length of the alignment after subtractionof the total number of gaps in the alignment. The percent identitydefined as herein can be obtained from NEEDLE by using the NOBRIEFoption and is labelled in the output of the program as“longest-identity”.

The polynucleotide and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the polynucleotide of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,(1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See the homepage of the NationalCenter for Biotechnology Information at http://www.ncbi.nlm.nih.gov/.

Vectors

Polynucleotides of the invention can be incorporated into a vector,including cloning and expression vectors. A vector may be a recombinantreplicable vector. The vector may be used to replicate a polynucleotideof the invention in a compatible host cell. The vector may convenientlybe subjected to recombinant DNA procedures

The invention also pertains to methods of growing, transforming ortransfecting such vectors in a suitable host cell, for example underconditions in which expression of a polypeptide of the invention occurs.The invention provides a method of making polypeptides of the inventionby introducing a polynucleotide of the invention into a vector, in anembodiment an expression vector, introducing the vector into acompatible host cell, and growing the host cell under conditions whichbring about replication of the vector. The vector may be recovered fromthe host cell.

A vector according to the invention may be an autonomously replicatingvector, i.e. a vector which exists as an extra-chromosomal entity, thereplication of which is independent of chromosomal replication, e.g. aplasmid. Alternatively, the vector may be one which, when introducedinto a host cell, is integrated into the host cell genome and replicatedtogether with the chromosome(s) into which it has been integrated.

One type of vector is a “plasmid”, which refers to a circular doublestranded DNA loop into which additional DNA segments can be inserted.Another type of vector is a viral vector, wherein additional DNAsegments can be inserted into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,bacterial integration vector with out a suitable origin of replicationor a non-episomal mammalian vectors) are integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome.

Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “expression vectors”. The terms “expression vector”,“expression construct” and “recombinant expression vector” are usedinterchangeably herein. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of plasmids. The terms“plasmid” and “vector can be used interchangeably herein as the plasmidis the most commonly used form of vector. However, the invention isintended to include such other forms of expression vectors, such ascosmid, viral vectors (e.g., replication defective retroviruses,adenoviruses and adeno-associated viruses) and phage vectors which serveequivalent functions.

Vectors according to the invention may be used in vitro, for example forthe production of RNA or used to transfect or transform a host cell, forexample a bacterial cell, and used for the production of analpha-amylase as encoded by a polynucleotide of the invention.

The recombinant expression vectors of the invention comprise apolynucleotide of the invention in a form suitable for expression of thepolynucleotide in a host cell, which means that the recombinantexpression vector includes one or more regulatory sequences, selected onthe basis of the host cells to be used for expression, which is operablylinked to the polynucleotide sequence to be expressed.

Within a recombinant expression vector, “operably linked” is intended tomean that the nucleotide sequence of interest is linked to theregulatory sequence(s) in a manner which allows for expression of thenucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell), i.e. the term “operably linked” refers to a juxtaposition whereinthe components described are in a relationship permitting them tofunction in their intended manner. A regulatory sequence such as apromoter, enhancer or other expression regulation signal “operablylinked” to a coding sequence is positioned in such a way that expressionof the coding sequence is achieved under conditions compatible with thecontrol sequences or the sequences are arranged so that they function inconcert for their intended purpose, for example transcription initiatesat a promoter and proceeds through the DNA sequence encoding thepolypeptide.

The term “regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignal). Such regulatory sequences are described, for example, inGoeddel; Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990).

The term regulatory sequences includes those sequences which directconstitutive expression of a nucleotide sequence in many types of hostcells and those which direct expression of the nucleotide sequence onlyin a certain host cell (e.g. tissue-specific regulatory sequences).

A vector or expression construct for a given host cell may thus comprisethe following elements operably linked to each other in a consecutiveorder from the 5′-end to 3′-end relative to the coding strand of thesequence encoding the polypeptide of the first invention: (1) a promotersequence capable of directing transcription of the nucleotide sequenceencoding the polypeptide in the given host cell; (2) a ribosome bindingsite to facilitate the translation of the transcribed RNA (3)optionally, a signal sequence capable of directing secretion of thepolypeptide from the given host cell into a culture medium; (4) apolynucleotide sequence according to the invention; and preferably also(5) a transcription termination region (terminator) capable ofterminating transcription downstream of the nucleotide sequence encodingthe polypeptide.

Downstream of the nucleotide sequence according to the invention theremay be a 3′ untranslated region containing one or more transcriptiontermination sites (e.g. a terminator, herein also referred to as a stopcodon). The origin of the terminator is less critical. The terminatorcan, for example, be native to the DNA sequence encoding thepolypeptide. However, preferably a bacterial terminator is used inbacterial host cells and a filamentous fungal terminator is used infilamentous fungal host cells. More preferably, the terminator isendogenous to the host cell (in which the nucleotide sequence encodingthe polypeptide is to be expressed). In the transcribed region, aribosome binding site for translation may be present. The coding portionof the mature transcripts expressed by the constructs will include astart codon is usually AUG (or ATG), but there are also alternativestart codons, such as for example GUG (or GTG) and UUG (or TTG), whichare used in prokaryotes. Also a stop or translation termination codon isappropriately positioned at the end of the polypeptide to be translated.

Enhanced expression of the polynucleotide of the invention may also beachieved by the selection of homologous and heterologous regulatoryregions, e.g. promoter, secretion leader and/or terminator regions,which may serve to increase expression and, if desired, secretion levelsof the protein of interest from the expression host and/or to providefor the inducible control of the expression of a polypeptide of theinvention.

It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired.The expression vectors of the invention can be introduced into hostcells to thereby produce proteins or peptides, encoded bypolynucleotides as described herein (e.g. the polypeptide having alphaamylase activity according to the invention or a variant thereof asdescribed herein).

The recombinant expression vectors of the invention, also referred toherein as “vector of the invention” can be designed for expression ofthe polypeptides according to the invention in prokaryotic or eukaryoticcells. For example, the polypeptides according to the invention can beproduced in bacterial cells such as E. coli and Bacilli, insect cells(using baculovirus expression vectors), fungal cells, yeast cells ormammalian cells. Suitable host cells are discussed herein and further inGoeddel, Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990). Alternatively, the recombinantexpression vector can be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.

For most bacteria, filamentous fungi and yeasts, the vector orexpression construct is preferably integrated in the genome of the hostcell in order to obtain stable transformants. In case the expressionconstructs are integrated in the host cells genome, the constructs areeither integrated at random loci in the genome, or at predeterminedtarget loci using homologous recombination, in which case the targetloci preferably comprise a highly expressed gene.

Accordingly, expression vectors useful in the present invention includechromosomal-, episomal- and virus-derived vectors e.g., vectors which isa plasmid, bacteriophage, yeast episome, yeast chromosomal elements,viruses such as baculoviruses, papova viruses, vaccinia viruses,adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses,and vectors which are combinations thereof, such as those consisting ofplasmid and bacteriophage genetic elements, such as cosmids andphagemids.

The polynucleotide according to the invention should be operativelylinked to an appropriate promoter. Aside from the promoter native to thegene encoding the polypeptide of the invention, other promoters may beused to direct expression of the polypeptide of the invention. Thepromoter may be selected for its efficiency in directing the expressionof the polypeptide of the invention in the desired expression host. Asuitable promoter may be one which is an “inducible promoter” is onewhich causes mRNA synthesis of a gene to be initiated temporally underspecific conditions. Alternatively, a promoter may be a “constitutive”promoter, i.e. one that permits the gene to be expressed under virtuallyall environmental conditions, i.e. a promoter that directs constant,non-specific gene expression. A “strong constitutive promoter”, i.e., apromoter which causes mRNAs to be initiated at high frequency comparedto a native host cell may be used.

In the invention, bacteria may preferably be used as host cells for theexpression of a polypeptide of the invention, in particular Bacilli.Suitable inducible promoters useful in such host cells include: (i)Promoters may be regulated primarily by an ancillary factor such as arepressor or an activator. The repressors are sequence-specific DNAbinding proteins that repress promoter activity. The transcription canbe initiated from this promoter in the presence of an inducer thatprevents binding of the repressor to the operator of the promoter.Examples of such promoters from Gram-positive microorganisms include,but are not limited to, gnt (gluconate operon promoter); penP fromBacillus licheniformis; glnA (glutamine synthetase); xylAB (xyloseoperon); araABD (L-arabinose operon) and P_(spac) promoter, a hybridSPO1/lac promoter that can be controlled by inducers such asisopropyl-β-D-thiogalactopyranoside [IPTG] ((Yansura D. G., Henner D. J.Proc Natl Acad Sci USA. 1984 81(2):439-443). Activators are alsosequence-specific DNA binding proteins that induce promoter activity.Examples of such promoters from Gram-positive microorganisms include,but are not limited to, two-component systems (PhoP-PhoR, DegU-DegS,SpoOA-Phosphorelay), LevR, Mry and GItC. (ii) Production of secondarysigma factors can be primarily responsible for the transcription fromspecific promoters. Examples from Gram-positive microorganisms include,but are not limited to, the promoters activated by sporulation specificsigma factors: σ^(F), σ^(E), σ^(G) and σ^(K) and general stress sigmafactor, σ^(B). The σ^(B)-mediated response is induced by energylimitation and environmental stresses (Hecker M, Volker U. MolMicrobiol. 1998; 29(5):1129-1136.). (iii) Attenuation andantitermination also regulates transcription. Examples fromGram-positive microorganisms include, but are not limited to, trp operonand sacB gene. (iv) Other regulated promoters in expression vectors arebased the sacR regulatory system conferring sucrose inducibility (KlierA F, Rapoport G. Annu Rev Microbiol. 1988; 42:65-95).

Strong constitutive promoters are well known and an appropriate one maybe selected according to the specific sequence to be controlled in thehost cell. Suitable inducible promoters useful in bacteria, such asBacilli, include: promoters from Gram-positive microorganisms such as,but are not limited to, SPO1-26, SPO1-15, veg, pyc (pyruvate carboxylasepromoter), and amyE. Examples of promoters from Gram-negativemicroorganisms include, but are not limited to, tac, tet, trp-tet, lpp,lac, lpp-lac, laclq, T7, T5, T3, gal, trc, ara, SP6, λ-P_(R), andλ-P_(L).

Additional examples of promoters useful in bacterial cells, such asBacilli, include the α-amylase and SPo2 promoters as well as promotersfrom extracellular protease genes.

In an embodiment, the promoter sequences may be obtained from abacterial source. In another embodiment, the promoter sequences may beobtained from a gram positive bacterium such as a Bacillus strain, e.g.,Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillusfirmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis,Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus,Bacillus subtilis, or Bacillus thuringiensis; or a Streptomyces strain,e.g., Streptomyces lividans or Streptomyces murinus; or from a gramnegative bacterium, e.g., E. coli or Pseudomonas sp.

An example of a suitable promoter for directing the transcription of apolynucleotide sequence in the methods of the present invention is thepromoter obtained from the E. coli lac operon. Another example is thepromoter of the Streptomyces coelicolor agarase gene (dagA). Anotherexample is the promoter of the Bacillus lentus alkaline protease gene(aprH). Another example is the promoter of the Bacillus licheniformisalkaline protease gene (subtilisin Carlsberg gene). Another example isthe promoter of the Bacillus subtilis levansucrase gene (sacB). Anotherexample is the promoter of the Bacillus subtilis alphaamylase gene(amyF). Another example is the promoter of the Bacillus licheniformisalphaamylase gene (amyL). Another example is the promoter of theBacillus stearothermophilus maltogenic amylase gene (amyM). Anotherexample is the promoter of the Bacillus amyloliquefaciens alpha-amylasegene (amyQ). Another example is a “consensus” promoter having thesequence TTGACA for the “−35” region and TATAAT for the “−10” region.Another example is the promoter of the Bacillus licheniformispenicillinase gene (penP). Another example are the promoters of theBacillus subtilis xylA and xylB genes.

A variety of promoters can be used that are capable of directingtranscription in the recombinant host cells of the invention. Preferablythe promoter sequence is from a highly expressed gene. Examples ofpreferred highly expressed genes from which promoters may be selectedand/or which are comprised in preferred predetermined target loci forintegration of expression constructs, include but are not limited togenes encoding glycolytic enzymes such as triose-phosphate isomerases(TPI), glyceraldehyde-phosphate dehydrogenases (GAPDH), phosphoglyceratekinases (PGK), pyruvate kinases (PYK or PKI), alcohol dehydrogenases(ADH), as well as genes encoding amylases, glucoamylases, proteases,xylanases, cellobiohydrolases, β-galactosidases, alcohol (methanol)oxidases, elongation factors and ribosomal proteins. Specific examplesof suitable highly expressed genes include e.g. the LAC4 gene fromKluyveromyces sp., the methanol oxidase genes (AOX and MOX) fromHansenula and Pichia, respectively, the glucoamylase (glaA) genes fromA. niger and A. awamori, the A. oryzae TAKA-amylase gene, the A.nidulans gpdA gene and the T. reesei cellobiohydrolase genes.

Examples of strong constitutive and/or inducible promoters which may beused in fungal expression host cells include those which are obtainablefrom the fungal genes for xylanase (xlnA), phytase, ATP-synthetase,subunit 9 (oliC), triose phosphate isomerase (tpi), alcoholdehydrogenase (AdhA), α-amylase (amy), amyloglucosidase (AG-from theglaA gene), acetamidase (amdS) and glyceraldehyde-3-phosphatedehydrogenase (gpd) promoters.

All of the above-mentioned promoters are readily available in the art.

The vector may contain a polynucleotide of the invention oriented in anantisense direction to provide for the production of antisense RNA.

Vector DNA can be introduced into prokaryotic or eukaryotic cells vianatural competence, conventional transformation or transfectiontechniques. As used herein, the terms “transformation” and“transfection” are intended to refer to a variety of art-recognizedtechniques for introducing foreign polynucleotide (e.g., DNA) into ahost cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, transduction,infection, lipofection, cationic lipidmediated transfection orelectroporation. Suitable methods for transforming or transfecting hostcells can be found in Sambrook, et al. (supra) and other laboratorymanuals.

In order to identify and select cells which harbour a vector, a genethat encodes a selectable marker (e.g., resistance to antibiotics) isgenerally introduced into the host cells along with the polynucleotideof the invention. Preferred selectable markers include, but are notlimited to, those which confer resistance to drugs or which complement adefect in the host cell.

Such markers include ATP synthetase, subunit 9 (oliC),orotidine-5′-phosphatedecarboxylase (pvrA), the bacterial G418resistance gene (this may also be used in yeast, but not in fungi), theampicillin resistance gene (E. coli), resistance genes for, neomycin,kanamycin, tetracycline, spectinomycin, erythromycin, chloramphenicol,phleomycin (Bacillus) and the E. coli uidA gene, coding forβ-glucuronidase (GUS). Vectors may be used in vitro, for example for theproduction of RNA or used to transfect or transform a host cell.

They also include e.g. versatile marker genes that can be used fortransformation of most filamentous fungi and yeasts such as acetamidasegenes or cDNAs (the amdS, niaD, facA genes or cDNAs from A. nidulans, A.oryzae or A. niger), or genes providing resistance to antibiotics likeG418, hygromycin, bleomycin, kanamycin, methotrexate, phleomycinorbenomyl resistance (benA). Alternatively, specific selection markerscan be used such as auxotrophic markers which require correspondingmutant host strains: e.g. D-alanine racemase (from Bacillus), URA3 (fromS. cerevisiae or analogous genes from other yeasts), pyrG or pyrA (fromA. nidulans or A. niger), argB (from A. nidulans or A. niger) or trpC.In an embodiment the selection marker is deleted from the transformedhost cell after introduction of the expression construct so as to obtaintransformed host cells capable of producing the polypeptide which arefree of selection marker genes.

Expression of proteins in prokaryotes is often carried out in withvectors containing constitutive or inducible promoters directing theexpression of either fusion or non-fusion proteins. Fusion vectors add anumber of amino acids to a protein encoded therein, e.g. to the aminoterminus of the recombinant protein. Such fusion vectors typically servethree purposes: 1) to increase expression of recombinant protein; 2) toincrease the solubility of the recombinant protein; and 3) to aid in thepurification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein.

Appropriate culture mediums and conditions for the above-described hostcells are known in the art.

Vectors preferred for use in bacteria are for example disclosed inWO-A1-2004/074468, which are hereby enclosed by reference. Othersuitable vectors will be readily apparent to the skilled artisan.

Vectors of the invention may be transformed into a suitable host cell asdescribed herein to provide for expression of a polypeptide of theinvention. Thus, in a further aspect the invention provides a processfor preparing a polypeptide according to the invention which comprisescultivating a host cell transformed or transfected with an expressionvector encoding the polypeptide, and recovering the expressedpolypeptide.

Host Cells

The invention further provides “recombinant host cells” also referredherein as “host cells” transformed or transfected with the vectors forthe replication and/or expression of polynucleotides of the invention.The cells will be chosen to be compatible with the said vector.Promoters and other expression regulation signals may be selected to becompatible with the host cell for which expression is designed.

The invention features cells, e.g., transformed host cells orrecombinant host cells comprising a polynucleotide according to theinvention or comprising a vector according to the invention.

A “transformed host cell” or “recombinant host cell” is a cell intowhich (or into an ancestor of which) has been introduced, by means ofrecombinant DNA techniques, a polynucleotide according to the invention.Both prokaryotic and eukaryotic cells are included, e.g., bacteria,fungi, yeast, insect, mammalian and the like.

Preferred are cells of a Bacillus strain, e.g., Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis; or a Streptomyces strain, e.g., Streptomyces lividans orStreptomyces murinus; or from a gram negative bacterium, e.g., E. colior Pseudomonas sp.

According to another aspect, the host cell is a eukaryotic host cell.Preferably, the eukaryotic cell is a mammalian, insect, plant, fungal,or algal cell. Preferred mammalian cells include e.g. Chinese hamsterovary (CHO) cells, COS cells, 293 cells, Per.C6® cells, and hybridomas.A number of vectors suitable for stable transfection of mammalian cellsare available to the public, methods for constructing such cell linesare also publicly known, e.g., in Ausubel et al. (supra).

In an embodiment insect cells include e.g. Sf9 and Sf21 cells andderivatives thereof.

In an embodiment the eukaryotic cell is a fungal cell, i.e. a yeastcell, such as Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia strain. Preferably from Kluyveromyceslactis, S. cerevisiae, Hansenula polymorpha, Yarrowia lipolytica andPichia pastoris, or a filamentous fungal cell.

Filamentous fungi include all filamentous forms of the subdivisionEumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworthand Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK). The filamentous fungiare characterized by a mycelial wall composed of chitin, cellulose,glucan, chitosan, mannan, and other complex polysaccharides. Vegetativegrowth is by hyphal elongation and carbon catabolism is obligatelyaerobic. Filamentous fungal strains include, but are not limited to,strains of Acremonium, Agaricus, Aspergillus, Aureobasidium,Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola,Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, andTrichoderma. In an embodiment filamentous fungal cells belong to aspecies of an Aspergillus, Chrysosporium, Penicillium, Talaromyces,Fusarium or Trichoderma genus, and preferably a species of Aspergillusniger, Aspergillus awamori, Aspergillus foetidus, Aspergillus sojae,Aspergillus fumigatus, Talaromyces emersonii, Aspergillus oryzae,Chrysosporium lucknowense, Myceliophthora thermophila, Fusariumoxysporum, Trichoderma reesei or Penicillium chrysogenum. In anembodiment the host cell is Aspergillus niger.

If the host cell according to the invention is an Aspergillus niger hostcell, the host cell preferably is CBS 513.88, CBS124.903 or a derivativethereof.

A host cell can be chosen that modulates the expression of the insertedsequences, or modifies and processes the gene product in a specific,desired fashion. Such modifications (e.g., glycosylation) and processing(e.g., cleavage) of protein products may facilitate optimal functioningof the protein.

Various host cells have characteristic and specific mechanisms forpost-translational processing and modification of proteins and geneproducts. Appropriate cell lines or host systems familiar to those ofskill in the art of molecular biology and/or microbiology can be chosento ensure the desired and correct modification and processing of theforeign protein produced. To this end, eukaryotic host cells thatpossess the cellular machinery for proper processing of the primarytranscript, glycosylation, and phosphorylation of the gene product canbe used. Such host cells are well known in the art.

If desired, a host cell as described above may be used to in thepreparation of a polypeptide according to the invention. Such a methodtypically comprises cultivating a recombinant host cell (e.g.transformed or transfected with an expression vector as described above)under conditions to provide for expression (by the vector) of a codingsequence encoding the polypeptide, and optionally recovering, morepreferably recovering and purifying the produced polypeptide from thecell or culture medium. Polynucleotides of the invention can beincorporated into a recombinant replicable vector, e.g. an expressionvector. The vector may be used to replicate the polynucleotide in acompatible host cell. Thus in a further embodiment, the inventionprovides a method of making a polynucleotide of the invention byintroducing a polynucleotide of the invention into a replicable vector,introducing the vector into a compatible host cell, and growing the hostcell under conditions which bring about the replication of the vector.The vector may be recovered from the host cell.

Preferably the polypeptide according to the invention is produced as asecreted protein in which case the nucleotide sequence encoding a matureform of the polypeptide in the expression construct is operably linkedto a nucleotide sequence encoding a signal sequence. Preferably thesignal sequence is native (homologous), also referred to herein as “wildtype” to the nucleotide sequence encoding the polypeptide. Alternativelythe signal sequence is foreign (heterologous) to the nucleotide sequenceencoding the polypeptide, in which case the signal sequence ispreferably endogenous to the host cell in which the nucleotide sequenceaccording to the invention is expressed. Examples of suitable signalsequences for Bacilli are from the amyE, yurI fliL, vpr, glpQ, phy,lytC, ywsB, ybbD, ybxI, yolA, ylqB, ybbC, peI, yckD, ywaD, ywmD, yweA,yraJ, dacF, yfjS, yybN, yrpD, yvcE, wprA, yxaL, ykwD, yncM2, sacB, phrC,SacC, yoqM, ykoJ, lip, yfkN, yurI, ybfO, yfkD, yoaJ, xynA, penP, ydjM,yddT, yojL, yomL, yqxI, yrvJ, yvpA, yjcM, yjfA, ypjP, ggt, yoqH, ywtD,ylaE, yraJ, lytB, lytD, nprB, nucB, rplR, yfhK, yjdB, ykvV, ybbE, yuiC,ylbL, yacD, yvpB genes from Bacillus subtilis. Suitable yeast signalsequences are those from yeast a-factor genes. Similarly, a suitablesignal sequence for filamentous fungal host cells is e.g. a signalsequence those from a filamentous fungal amyloglucosidase (AG) gene,e.g. the A. niger glaA gene. This may be used in combination with theamyloglucosidase (also called (gluco) amylase) promoter itself, as wellas in combination with other promoters. Hybrid signal sequences may alsobe used with the context of the present invention.

Preferred heterologous secretion leader sequences are those originatingfrom the fungal amyloglucosidase (AG) gene (glaA-both 18 and 24 aminoacid versions e.g. from Aspergillus), the α-factor gene (yeasts e.g.Saccharomyces and Kluyveromyces) or the α-amylase (amyE, amyQ and amyL)and alkaline protease aprE and natural protease genes (Bacillus). Thevectors may be transformed or transfected into a suitable host cell asdescribed above to provide for expression of a polypeptide of theinvention. This process may comprise culturing a host cell transformedwith an expression vector as described above under conditions to providefor expression by the vector of a coding sequence encoding thepolypeptide.

The invention thus provides host cells transformed or transfected withor comprising a polynucleotide or vector of the invention. Preferablythe polynucleotide is carried in a vector for the replication andexpression of the polynucleotide. The cells will be chosen to becompatible with the said vector and may for example be prokaryotic (forexample bacterial), fungal, yeast or plant cells.

A heterologous host cell may also be chosen wherein the polypeptide ofthe invention is produced in a form which is substantially free ofenzymatic activities that might interfere with the applications, e.g.free from starch degrading, cellulose-degrading or hemicellulosedegrading enzymes. This may be achieved by choosing a host cell whichdoes not normally produce such enzymes.

The invention encompasses processes for the production of thepolypeptide of the invention by means of recombinant expression of a DNAsequence encoding the polypeptide. For this purpose the DNA sequence ofthe invention can be used for gene amplification and/or exchange ofexpression signals, such as promoters, secretion signal sequences, inorder to allow economic production of the polypeptide in a suitablehomologous or heterologous host cell. A homologous host cell is a hostcell which is of the same species or which is a variant within the samespecies as the species from which the DNA sequence is obtained.

The host cell may over-express the polypeptide, and techniques forengineering over-expression are well known. The host may thus have twoor more copies of the encoding polynucleotide (and the vector may thushave two or more copies accordingly).

Therefore in one embodiment of the invention the recombinant host cellaccording to the invention is capable of expressing or overexpressing apolynucleotide or vector according to the invention.

Another aspect of the invention is a method for producing a polypeptideof the invention comprising (a) culturing a recombinant host cellaccording to the invention under conditions such that the polypeptide ofthe invention is produced; and (b) optionally recovering the polypeptideof the invention from the cell culture medium.

According to the present invention, the production of the polypeptide ofthe invention can be effected by the culturing of a host cell accordingto the invention, which has been transformed with one or morepolynucleotides of the present invention, in a conventional nutrientfermentation medium. The method of the invention comprises the step ofculturing a host cell of the invention under conditions such that apolypeptide of the invention is produced.

The recombinant host cells according to the invention may be culturedusing procedures known in the art. For each combination of a promoterand a host cell, culture conditions are available which are conducive tothe expression the DNA sequence encoding the polypeptide. After reachingthe desired cell density or titre of the polypeptide the culture isstopped and the polypeptide is recovered using known procedures.

The term “culturing” includes maintaining and/or growing a livingrecombinant host cell of the present invention, in particular therecombinant host cell according to the invention. In one aspect, arecombinant host cell of the invention is cultured in liquid media. Inanother aspect, a recombinant host cell is cultured in solid media orsemi-solid media.

Preferably, the recombinant host cell of the invention is cultured inliquid media comprising nutrients essential or beneficial to themaintenance and/or growth of the recombinant host cell. Such nutrientsinclude, but are not limited to, carbon sources or carbon substrates,e.g. complex carbohydrates such as bean or grain meal, starches, sugars,sugar alcohols, hydrocarbons, oils, fats, fatty acids, organic acids andalcohols; nitrogen sources, e.g. vegetable proteins, peptones, peptidesand amino acids obtained from grains, beans and tubers, proteins,peptides and amino acids obtained from animal sources such as meat, milkand animal byproducts such as peptones, meat extracts and caseinhydrolysates; inorganic nitrogen sources such as urea, ammonium sulfate,ammonium chloride, ammonium nitrate and ammonium phosphate; phosphoroussources, e.g. phosphoric acid, sodium and potassium salts thereof; traceelements, e.g. magnesium, iron, manganese, calcium, copper, zinc, boron,molybdenum and/or cobalt salts; as well as growth factors such as aminoacids, vitamins, growth promoters and the like.

The selection of the appropriate medium may be based on the choice ofexpression host, i.e. the choice of the recombinant host cell and/orbased on the regulatory requirements of the expression construct. Suchmedia are known to those skilled in the art. The medium may, if desired,contain additional components favouring the transformed expression hostsover other potentially contaminating microorganisms.

The recombinant host cells may be cultured in liquid media eithercontinuously or intermittently, by conventional culturing methods suchas standing culture, test tube culture, shaking culture, aerationspinner culture or fermentation. Preferably, the recombinant host cellsare cultured in a fermentor. Fermentation processes of the inventioninclude batch, fed-batch and continuous methods of fermentation. Avariety of such processes have been developed and are well known in theart.

The recombinant host cells are preferably cultured under controlled pH.In one embodiment, recombinant host cells may be cultured at a pH ofbetween 4.5 and 8.5, preferably 6.0 and 8.5, more preferably at a pH ofabout 7. The desired pH may be maintained by any method known to thoseskilled in the art.

Preferably, the recombinant host cells are further cultured undercontrolled aeration and under controlled temperatures. In oneembodiment, the controlled temperatures include temperatures between 15and 70° C., preferably the temperatures are between 20 and 55° C., morepreferably between 30 and 50° C.

The appropriate conditions are usually selected based on the choice ofthe expression host and the protein to be produced.

After fermentation, if necessary, the cells can be removed from thefermentation broth by means of centrifugation or filtration. Afterfermentation has stopped or after removal of the cells, the polypeptideof the invention may then be recovered and, if desired, purified andisolated by conventional means, including, but not limited to, treatmentwith a conventional resin, treatment with a conventional adsorbent,alteration of pH, solvent extraction, dialysis, filtration,concentration, crystallization, recrystallization, pH adjustment,lyophilisation and the like.

For example, the alpha-amylase enzyme according to the invention can berecovered and purified from recombinant cell cultures by methods knownin the art (Protein Purification Protocols, Methods in Molecular Biologyseries by Paul Cutler, Humana Press, 2004).

Usually, the compound is “isolated” when the resulting preparation issubstantially free of other components. In one embodiment, thepreparation has a purity of greater than about 80% (by dry weight) ofthe desired compound (e.g. less than about 20% of all the media,components or fermentation byproducts), in another embodiment greaterthan about 90% of the desired compound, in another embodiment greaterthan about 95% of the desired compound and in another embodiment greaterthan about 98 to 99% of the desired compound.

Alternatively, however, the desired compound is not purified from therecombinant host cell or the culture. The entire culture or the culturesupernatant may be used as a source of the product. In a specificembodiment, the culture or the culture supernatant is used withoutmodification. In a further embodiment, the culture or the culturesupernatant is concentrated, dried and/or lyophilized.

The recombinant host cell of the invention is capable of producing apolypeptide of the invention compound under suitable conditions.Preferably, production of a polypeptide of the invention meansproduction of at least about 50 mg, about 100 mg, about 200 mg, about500 mg, about 1 g, about 3 g, about 5 g or about 10 g polypeptide of theinvention per litre culture medium.

Enzyme Preparation

Bacillus strain DSM-AMB154-1 (see examples) was cultivated under aerobicconditions in a suitable fermentation medium.

A suitable medium medium may contain assimilable sources of carbon andnitrogen besides inorganic salts optionally together with growthpromoting nutrients, such as yeast extract. Fermentation is typicallyconducted at 35-40° C. and at a pH of 6.5-7.5 and preferably keptapproximately constant by automatic means. The enzyme is excreted intothe medium. At the end of fermentation, if required, the production hostmay be killed by means known by the person skilled in the art. Theensuing fermentation broth may be freed of bacterial cells, debristherefrom together with other solids, for example by filtration orcentrifugation. The filtrate or supernatant containing the enzyme may befurther clarified, for example by filtration or centrifugation, and thenconcentrated as required, for example by ultrafiltration or in anevaporator under reduced pressure to give a concentrate which, ifdesired, may be taken to dryness, for example by lyophilization orspray-drying. Typically, the resulting crude enzyme product exhibits anactivity in the range of about 10,000-500,000 MU per gram.

The polynucleotide according to the invention comprises a nucleotidesequence selected from:

The invention relates to a polynucleotide encoding for a polypeptidehaving alpha-amylase activity comprising:

-   -   (a) a polynucleotide sequence encoding a polypeptide having an        amino acid sequence as set out in SEQ ID NO: 2 or having an        amino acid sequence as set out in amino acids 34 to 719 of SEQ        ID NO: 2; or    -   (b) a polynucleotide sequence encoding a polypeptide having at        least 99.5% identity to a polypeptide having an amino acid        sequence as set out in amino acids 34 to 719 of SEQ ID NO: 2; or    -   (c) a polynucleotide sequence as set out in nucleotides 100 to        2157 of SEQ ID NO: 1 or SEQ ID NO: 3; or    -   (d) a polynucleotide sequence as set out in SEQ ID NO: 1 or SEQ        ID NO: 3.

The polynucleotide according to the invention encodes for analpha-amylase.

In an embodiment of the polynucleotide according to the invention, thepolynucleotide is an isolated polynucleotide comprising:

-   -   (a) a polynucleotide sequence as set out in SEQ ID NO: 1 or 3;        or    -   (b) a polynucleotide sequence as set out in nucleotides 100 to        2157 of the polynucleotide sequence of SEQ ID NO: 1 or 3        (inclusive of nucleotides 100 and 2157, for the avoidance of        doubt); or    -   (c) a polynucleotide sequence encoding a polypeptide having an        amino acid sequence as set out in SEQ ID NO: 2 or having an        amino acid sequence as set out in amino acids 34 to 719 of SEQ        ID NO: 2 (inclusive of amino acids 34 and 719, for the avoidance        of doubt).

In an embodiment of the polynucleotide according to the invention, thepolynucleotide is produced by Alicyclobacillus pohliae NCIMB14276.

In a further aspect of the polynucleotide according to the invention theisolated polynucleotide is produced by Alicyclobacillus pohliaeNCIMB14276.

The vector according to the invention comprises the polynucleotidesequence according to the invention.

In an embodiment of the vector according to the invention the vector isan expression vector, wherein the polynucleotide sequence according tothe invention is operably linked with at least one regulatory sequenceallowing for expression of the polynucleotide sequence in a suitablehost cell.

Suitable host cells include bacteria, including Escherichia, Anabaena,Caulobactert, Gluconobacter, Rhodobacter, Pseudomonas, Paracoccus,Bacillus, Brevibacterium, Corynebacterium, Rhizobium (Sinorhizobium),Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus,Methylobacterium, Staphylococcus or Streptomyces. In an aspect of thevector according to the invention, the host cell is a the bacterial cellis selected from the group consisting of B. subtilis, B. puntis, B.megaterium, B. halodurans, B. pumilus, G. oxydans, Caulobactertcrescentus CB 15, Methylobacterium extorquens, Rhodobacter sphaeroides,Pseudomonas zeaxanthinifaciens, Paracoccus denitrificans, C. glutamicum,Staphylococcus carnosus, Streptomyces lividans, Sinorhizobium meliotiand Rhizobium radiobacter.

In a further embodiment of the vector according to the invention thesuitable host cell is a is an Aspergillus, Bacillus, Chrysosporium,Escherichia, Kluyveromyces, Penicillium, Pseudomonas, Saccharomyces,Streptomyces or Talaromyces species, preferably the host cell is aBacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis,Escherichia coli, Aspergillus Niger or Aspergillus oryzae species.

The recombinant host cell according to the invention may comprise thepolynucleotide according to the invention or the vector according to theinvention.

In an embodiment of the recombinant host cell according the invention,the recombinant host cell is capable of expressing or over-expressingthe polynucleotide according to the invention or the vector according tothe invention.

The method according to the invention for manufacturing thepolynucleotide according to the invention or the vector according to theinvention comprises the steps of culturing a host cell transformed withsaid polynucleotide or said vector and isolating said polynucleotide orsaid vector from said host cell.

The polypeptide according to the invention comprises:

-   an alpha-amylase polypeptide comprising:    -   (a) an amino acid sequence as set out in amino acids 34 to 719        of SEQ ID NO: 2; or    -   (b) an amino acid sequence having at least 99.5% identity to an        amino acid sequence as set out in amino acids 34 to 719 of SEQ        ID NO: 2; or    -   (c) an amino acid sequence encoded by a polynucleotide as set        out in nucleotides 100 to 2157 of SEQ ID NO: 1 or SEQ ID NO: 3;        or    -   (d) an amino acid sequence having at least 70% identity to an        amino acid sequence as set out in amino acids 34 to 719 of SEQ        ID NO: 2 and having at least one of Asp at position 184, Ala at        position 297, Thr at position 368 and Asn at position 489, said        positions being defined with reference to SEQ ID NO: 2; or    -   (e) an amino acid sequence having at least 70% identity to an        amino acid sequence as set out in amino acids 34 to 719 of SEQ        ID NO: 2 and having at least one of Asp at position 184, Ala at        position 297, Thr at position 368 and Asn at position 489, said        positions being defined with reference to SEQ ID NO: 2 and said        amino acid sequence characterized in that when used to prepare a        baked product having a least 5 wt % sugar based on flour, said        baked product has reduced hardness after storage in comparison        with a baked product prepared without use of said amino acid        sequence.

In an embodiment of the polypeptide according to the invention, thepolypeptide is an isolated polypeptide comprising:

-   -   (a) amino acid sequence as set out in amino acids 34 to 719 of        SEQ ID NO: 2; or    -   (b) an amino acid sequence encoded by the polynucleotide as set        out in nucleotides 100 to 2157 of SEQ ID NO: 1 or 3; or    -   (c) the amino acid sequence according to (b), wherein the        polynucleotide is produced by Alicyclobacillus pohliae        NCIMB14276.

In an embodiment of the polypeptide according to the invention, thepolypeptide is obtainable by expressing the polynucleotide according tothe invention or the vector according to the invention in an appropriatehost cell.

The method according to the invention for manufacturing the polypeptideaccording the invention comprises cultivating the recombinant host cellaccording to the invention under condition which allow for expression ofthe polynucleotide according to the invention or the vector according tothe invention and, optionally, recovering the encoded polypeptide fromthe cell or culture medium.

In an embodiment of the method according to the invention formanufacturing the polypeptide according to the invention the methodcomprises cultivating a host cell comprising a vector, the vectorcomprising a polynucleotide comprising:

-   -   (a) a polynucleotide sequence as set out in SEQ ID NO: 1 or 3;        or    -   (b) a polynucleotide sequence as set out in nucleotides 100 to        2157 of the polynucleotide sequence of SEQ ID NO: 1 or 3        (inclusive of nucleotides 100 and 2157, for the avoidance of        doubt); or    -   (c) a polynucleotide sequence encoding a polypeptide having an        amino acid sequence as set out in SEQ ID NO: 2 or having an        amino acid sequence as set out in amino acids 34 to 719 of SEQ        ID NO: 2 (inclusive of amino acids 34 and 719, for the avoidance        of doubt),        under conditions which allow for expression of the vector and,        optionally, recovering the encoded polypeptide from the cell or        culture medium.

In an embodiment of the method according to the invention formanufacturing the polypeptide according to the invention the methodcomprises cultivating a host cell comprising a polynucleotide, saidpolynucleotide comprising:

-   -   (a) a polynucleotide sequence as set out in SEQ ID NO: 1 or 3;        or    -   (b) a polynucleotide sequence as set out in nucleotides 100 to        2157 of the polynucleotide sequence of SEQ ID NO: 1 or 3        (inclusive of nucleotides 100 and 2157, for the avoidance of        doubt); or    -   (c) a polynucleotide sequence encoding a polypeptide having an        amino acid sequence as set out in SEQ ID NO: 2 or having an        amino acid sequence as set out in amino acids 34 to 719 of SEQ        ID NO: 2 (inclusive of amino acids 34 and 719, for the avoidance        of doubt),    -   under conditions which allow for expression of the        polynucleotide and, optionally, recovering the encoded        polypeptide from the cell or culture medium.

The polypeptide according to the invention may be used in foodmanufacturing.

In an embodiment of the use in food manufacturing, the use is themanufacture of a baked product, including without limitation a bread ora cake.

The enzyme composition according to the invention comprises thepolypeptide according to the invention and one or more componentsselected from the group consisting of milk powder, gluten, granulatedfat, an additional enzyme, an amino acid, a salt, oxidants (includingascorbic acid, bromate and Azodicarbonamide (ADA)), reducing agents(including L-cysteine), emulsifiers (including mono/di glycerides,monoglycerides such as glycerol monostearate (GMS), sodium stearoyllactylate (SSL), calcium stearoyl lactylate (CSL), polyglycerol estersof fatty acids (PGE) and diacetyl tartaric acid esters of mono- anddiglycerides (DATEM), gums (including guargum and xanthangum), flavours,acids (including citric acid, propionic acid), starch, modified starch,gluten, humectants (including glycerol) and preservatives.

In an embodiment of the enzyme composition according to the inventionthe additional enzyme is a lipolytic enzyme, preferably a phospholipase,a galactolipase or an enzyme having both phospholipase and galactolipaseactivity.

In an embodiment of the enzyme composition according to the inventionthe additional enzyme is a phospholipase.

In an embodiment of the enzyme composition according to the inventionthe additional enzyme is a galactolipase.

In an embodiment of the enzyme composition according to the inventionthe additional enzyme is an enzyme having both phospholipase andgalactolipase activity.

The method according to the invention to prepare a dough comprises thestep of combining the polypeptide according to the invention or theenzyme composition according to the invention and at least one doughingredient. ‘Combining’ includes without limitation, adding thepolypeptide or the enzyme composition according to the invention to theat least one dough ingredient, adding the at least one dough ingredientto the polypeptide or the enzyme composition according to the invention,mixing the polypeptide according to the invention and the at least onedough ingredient.

A dough ingredient includes a component selected from flour, egg, water,salt, sugar, flavours, fat (including butter, margarine, oil andshortening), baker's yeast, a chemical leavening system, milk, oxidants(including ascorbic acid, bromate and Azodicarbonamide (ADA)), reducingagents (including L-cysteine), emulsifiers (including mono/diglycerides, mono glycerides such as glycerol monostearate (GMS), sodiumstearoyl lactylate (SSL), calcium stearoyl lactylate (CSL), polyglycerolesters of fatty acids (PGE) and diacetyl tartaric acid esters of mono-and diglycerides (DATEM), gums (including guargum and xanthangum), acids(including citric acid, propionic acid), starch, modified starch,gluten, humectants (including glycerol) and preservatives.

In an aspect of the method according to the invention to prepare adough, the method comprises the steps of combining the polypeptideaccording to the invention and at least one component selected fromflour, egg, water, salt, sugar, flavours, fat (including butter,margarine, oil and shortening), baker's yeast, a chemical leaveningsystems, milk, oxidants (including ascorbic acid, bromate andAzodicarbonamide (ADA)), reducing agents (including L-cysteine),emulsifiers (including mono/di glycerides, monoglycerides such asglycerol monostearate (GMS), sodium stearoyl lactylate (SSL), calciumstearoyl lactylate (CSL), polyglycerol esters of fatty acids (PGE) anddiacetyl tartaric acid esters of mono- and diglycerides (DATEM), gums(including guargum and xanthangum), acids (including citric acid,propionic acid), starch, modified starch, gluten, humectants (includingglycerol) and preservatives.

In an aspect of the method according to the invention to prepare adough, the method comprises the steps of combining the enzymecomposition according to the invention and at least one componentselected from flour, egg, water, salt, sugar, flavours, fat (includingbutter, margarine, oil and shortening), baker's yeast, a chemicalleavening systems, milk, oxidants (including ascorbic acid, bromate andAzodicarbonamide (ADA)), reducing agents (including L-cysteine),emulsifiers (including mono/di glycerides, monoglycerides such asglycerol monostearate (GMS), sodium stearoyl lactylate (SSL), calciumstearoyl lactylate (CSL), polyglycerol esters of fatty acids (PGE) anddiacetyl tartaric acid esters of mono- and diglycerides (DATEM), gums(including guargum and xanthangum), acids (including citric acid,propionic acid), starch, modified starch, gluten, humectants (includingglycerol) and preservatives.

‘Combining’ in the above two aspects includes without limitation, addingthe polypeptide or the enzyme composition according to the invention tothe at least one component indicated above, adding the at least onecomponent indicated above to the polypeptide or the enzyme compositionaccording to the invention, mixing the polypeptide according to theinvention and the at least one component indicated above.

A dough according to the invention may comprise the polypeptideaccording to the invention or the enzyme composition according to theinvention.

The method according to the invention to prepare a baked productcomprises the step of baking the dough according to the invention.

In an embodiment of the method to prepare a baked product, the methodcomprises baking a dough comprising the polypeptide according to theinvention.

In an embodiment of the method to prepare a baked product, the methodcomprises baking a dough comprising the enzyme composition according tothe invention.

In an embodiment of the method to prepare a baked product the bakedproduct is bread or cake.

The baked product according to the invention is obtainable by the methodaccording to the invention to prepare the baked product.

The method according the invention to produce a polypeptide having atleast 60% identity, in an embodiment at least 70% identity, in anembodiment at least 80% identity, in an embodiment at least 85%identity, in an embodiment at least 90% identity, in an embodiment atleast 95% identity with

-   -   (a) a polypeptide having an amino acid sequence as set out in        amino acids 34 to 719 of SEQ ID NO: 2; or    -   (b) a polypeptide having at least 99.5% identity to a        polypeptide having an amino acid sequence as set out in amino        acids 34 to 719 of SEQ ID NO: 2; or    -   (c) an amino acid sequence encoded by the polynucleotide as set        out in nucleotides 100 to 2157 of SEQ ID NO: 1 or 3, comprises        the use of Alicyclobacillus pohliae NCIMB14276.

The alpha-amylase according to the invention is a starch degradingenzyme. Alpha-amylase activity can suitably be determined using theCeralpha® procedure, which is recommend by the American Association ofCereal Chemists (AACC).

A lipolytic enzyme, also referred to herein as lipase, is an enzyme thathydrolyses triacylglycerol and/or galactolipid and or phospholipids.

Lipase activity may be determined spectrophotometrically by using thechromogenic substrate p-nitrophenyl palmitate (pNPP, Sigma N-2752). Inthis assay the pNPP is dissolved in 2-propanol (40 mg pNPP per 10 ml2-propanol (Merck 1.09634)) and suspended in 100 mM Acetate bufferpH=5.0 containing 1.0% Triton X-100 (Merck 1.12298) (5 ml substrate in45 ml buffer). The final substrate concentration is 1.1 mM. The lipaseis incubated with this substrate solution at 37° C. for 10 minutes. Thereaction is stopped by addition of stop buffer 2% TRIS (Merck 1.08387)+1% Triton X-100 in a 1:1 ratio with respect to the reaction mixture andsubsequently the formed p-nitrophenol (pNP) is measured at 405 nm. Thisassay can also be applied at different pH values in order to determinepH dependence of a lipase. It should be understood that at different pHvalues different buffers might be required or that different detergentsmight be necessary to emulsify the substrate. One lipase unit is definedas the amount of enzyme that liberates 1 micromole of p-nitrophenol perminute at the reaction conditions stated. It should be understood thatit is not uncommon practice in routine analysis to use standardcalibration enzyme solutions with known activity determined in adifferent assay to correlate activity a given assay with units as wouldbe determined in the calibration assay.

Alternatively, lipase activity may be determined by using2,3-mercapto-1-propanol-tributyrate (TBDMP) as a substrate. Lipasehydrolyses the thioester bond(s) of TBDMP thereby liberating butanoicacid and 2,3-mercapto-1-propanol-dibutyrate,2,3-mercapto-1-propanol-monobutyrate or 2,3-mercapto-1-propanol. Theliberated thiol groups are titrated in a subsequent reaction with4,4,-dithiodipyridine (DTDP) forming 4-thiopyridone. The latter is in atautomeric equilibrium with 4-mercapthopyridine which absorbs at 334 nm.The reaction is carried out in 0.1 M acetate buffer pH 5.0 containing0.2% Triton-X100, 0.65 mM TBDMP and 0.2 mM DTDP at 37° C. One lipaseunit is defined as the amount of enzyme that liberates 1 micromole of4-thiopyridone per minute at the reaction conditions stated.

In addition to spectrophotometric measurement lipase activity may alsobe determined using titrimetric measurement. For example the esteraseactivity of a lipolytic enzyme may be measured on tributyrin as asubstrate according to Food Chemical Codex, Forth Edition, NationalAcademy Press, 1996, p 803.

A phospholipase is an enzyme that catalyzes the release of fatty acylgroups from a phospholipid. It may be a phospholipase A2 (PLA2, EC3.1.1.4) or a phospholipase A1 (EC 3.1.1.32). It may or may not haveother activities such as triacylglycerol lipase (EC 3.1.1.3) and/orgalactolipase (EC 3.1.1.26) activity.

The phospholipase may be a native enzyme from mammalian or microbialsources. An example of a mammalian phospholipase is pancreatic PLA2,e.g. bovine or porcine PLA2 such as the commercial product Lecitase 10L(porcine PLA2, product of Novozymes A/S).

Microbial phospholipases may be from Fusarium, e.g. F. oxysporumphospholipase A1 WO 1998/026057), F. venenatum phospholipase A1(described in WO 2004/097012 as a phospholipase A2 called FvPLA2), fromTuber, e.g. T. borchii phospholipase A2 (called TbPLA2, WO 2004/097012).

The phospholipase may also be a lipolytic enzyme variant withphospholipase activity, e.g. as described in WO 2000/032758 or WO2003/060112.

The phospholipase may also catalyze the release of fatty acyl groupsfrom other lipids present in the dough, particularly wheat lipids. Thus,the phospholipase may have triacylglycerol lipase activity (EC 3.1.1.3)and/or galactolipase activity (EC 3.1.1.26).

The phospholipase may be a lipolytic enzyme as described inWO2009/106575, such as the commercial product Panamore®, product of DSM.

The term ‘baked product’ refers to a baked food product prepared from adough. Examples of baked products, whether of a white, brown orwhole-meal type, which may be advantageously produced by the presentinvention include bread (in particular white, whole-meal or rye bread),typically in the form of loaves or rolls, French baguette-type bread,pastries, croissants, brioche, panettone, pasta, noodles (boiled or(stir-)fried), pita bread and other flat breads, tortillas, tacos,cakes, pancakes, cookies in particular biscuits, doughnuts, includingyeasted doughnuts, bagels, pie crusts, steamed bread, crisp bread,brownies, sheet cakes, snack foods (e.g., pretzels, tortilla chips,fabricated snacks, fabricated potato crisps). The term baked productincludes, bread containing from 2 to 30 wt % sugar, fruit containingbread, breakfast cereals, cereal bars, eggless cake, soft rolls andgluten-free bread. Gluten free bread herein and herein after is breadthan contains at most 20 ppm gluten. Several grains and starch sourcesare considered acceptable for a gluten-free diet. Frequently usedsources are potatoes, rice and tapioca (derived from cassava) Bakedproduct includes without limitation tin bread, loaves of bread, twists,buns, such as hamburger buns or steamed buns, chapati, rusk, dried steambun slice, bread crumb, matzos, focaccia, melba toast, zwieback,croutons, soft pretzels, soft and hard bread, bread sticks, yeastleavened and chemically-leavened bread, laminated dough products such asDanish pastry, croissants or puff pastry products, muffins, danish,bagels, confectionery coatings, crackers, wafers, pizza crusts,tortillas, pasta products, crepes, waffles, parbaked products andrefrigerated and frozen dough products.

An example of a parbaked product includes, without limitation, partiallybaked bread that is completed at point of sale or consumption with ashort second baking process.

The bread may be white or brown pan bread; such bread may for example bemanufactured using a so called American style Sponge and Dough method oran American style Direct method.

The term tortilla herein includes corn tortilla and wheat tortilla. Acorn tortilla is a type of thin, flat bread, usually unleavened madefrom finely ground maize (usually called “corn” in the United States). Aflour tortilla is a type of thin, flat bread, usually unleavened, madefrom finely ground wheat flour. The term tortilla further includes asimilar bread from South America called arepa, though arepas aretypically much thicker than tortillas. The term tortilla furtherincludes a laobing, a pizza-shaped thick “pancake” from China and anIndian Roti, which is made essentially from wheat flour. A tortillausually has a round or oval shape and may vary in diameter from about 6to over 30 cm.

The term “dough” is defined herein as a mixture of flour and otheringredients. In one aspect the dough is firm enough to knead or roll.The dough may be fresh, frozen, prepared or parbaked. The preparation offrozen dough is described by Kulp and Lorenz in Frozen and RefrigeratedDoughs and Batters.

Dough is made using dough ingredients, which include without limitation(cereal) flour, a lecithin source including egg, water, salt, sugar,flavours, a fat source including butter, margarine, oil and shortening,baker's yeast, chemical leavening systems such as a combination of anacid (generating compound) and bicarbonate, a protein source includingmilk, soy flour, oxidants (including ascorbic acid, bromate andAzodicarbonamide (ADA)), reducing agents (including L-cysteine),emulsifiers (including mono/di glycerides, monoglycerides such asglycerol monostearate (GMS), sodium stearoyl lactylate (SSL), calciumstearoyl lactylate (CSL), polyglycerol esters of fatty acids (PGE) anddiacetyl tartaric acid esters of mono- and diglycerides (DATEM), gums(including guargum and xanthangum), flavours, acids (including citricacid, propionic acid), starch, modified starch, gluten, humectants(including glycerol) and preservatives.

Cereals include maize, rice, wheat, barley, sorghum, millet, oats, rye,triticale, buckwheat, quinoa, spelt, einkorn, emmer, durum and kamut.

Dough is usually made from basic dough ingredients including (cereal)flour, such as wheat flower or rice flour, water and optionally salt.For leavened products, primarily baker's yeast is used next to chemicalleavening systems such as a combination of an acid (generating compound)and bicarbonate.

The term dough herein includes a batter. A batter is a semi-liquidmixture, being thin enough to drop or poor from a spoon, of one or moreflours combined with liquids such as water, milk or eggs used to preparevarious foods, including cake.

The dough may be made using a mix including a cake mix, a biscuit mix, abrownie mix, a bread mix, a pancake mix and a crepe mix.

The term dough includes frozen dough, which may also be referred to asrefrigerated dough. There are different types of frozen dough; thatwhich is frozen before proofing and that which is frozen after a partialor complete proofing stage. The frozen dough is typically used formanufacturing baked products including without limitation biscuits,breads, bread sticks and croissants.

The invention also relates to the use of the alpha-amylase according tothe invention in a number of industrial processes. Despite the long-termexperience obtained with these processes, the alpha-amylase according tothe invention may feature advantages over the enzymes currently used.Depending on the specific application, these advantages may includeaspects like lower production costs, higher specificity towards thesubstrate, less antigenic, less undesirable side activities, higheryields when produced in a suitable microorganism, more suitable pH andtemperature ranges, better tastes of the final product as well as foodgrade and kosher aspects.

In an embodiment the alpha-amylase according to the invention may beused in the food industry, including in food manufacturing.

An example of an industrial application of the alpha-amylase enzymeaccording to the invention in food is its use in baking applications.The alpha-amylase according to the invention may for example be used inbaked products such as bread or cake. For example to improve quality ofthe dough and/or the baked product.

Therefore in one embodiment of the invention provides the use of thealpha-amylase according to the invention in the preparation of a doughand provides a dough comprising the alpha-amylase according to theinvention. The invention also provides the preparation of a doughcomprising the steps of adding the alpha-amylase according to theinvention to at least one dough ingredient.

Yeast, enzymes and optionally additives are generally added separatelyto the dough.

Enzymes may be added in a dry, e.g. granulated form, in a liquid form orin the form of a paste. Additives are in most cases added in powderform. Suitable additives include oxidants (including ascorbic acid,bromate and Azodicarbonamide (ADA)), reducing agents (includingL-cysteine), emulsifiers (including mono/di glycerides, monoglyceridessuch as glycerol monostearate (GMS), sodium stearoyl lactylate (SSL),calcium stearoyl lactylate (CSL), polyglycerol esters of fatty acids(PGE) and diacetyl tartaric acid esters of mono- and diglycerides(DATEM), gums (including guargum and xanthangum), flavours, acids(including citric acid, propionic acid), starch, modified starch,gluten, humectants (including glycerol) and preservatives.

The preparation of a dough from the dough ingredients is well known inthe art and includes mixing of said ingredients and optionally one ormore moulding and fermentation steps.

The preparation of baked products from such doughs is also well known inthe art and may comprise moulding and shaping and further fermentationof the dough followed by baking at required temperatures and bakingtimes. In one embodiment the invention provides a method to prepare abaked product comprising the step of baking the dough according to theinvention. The baking of the dough to produce a baked product may beperformed using methods well known in the art. The invention alsoprovides a baked product obtainable according to this method. In anembodiment the baked product according to the invention is bread orcake. In one aspect of the invention, the alpha-amylase according to theinvention may be used to prepare laminated doughs for baked productswith improved crispiness.

The present invention also relates to methods for preparing a dough or abaked product comprising incorporating into the dough an effectiveamount of the alpha-amylase according to the invention, which improvesone or more properties of the dough or the baked product obtained fromthe dough relative to a dough or a baked product in which thepolypeptide is not incorporated.

The phrase “incorporating into the dough” is defined herein as addingthe alpha-amylase enzyme according to the invention to the dough, anyingredient from which the dough is to be made, and/or any mixture ofdough ingredients from which the dough is to be made. In other words,the alpha-amylase enzyme according to the invention may be added in anystep of the dough preparation and may be added in one, two or moresteps. The alpha-amylase enzyme according to the invention is added tothe ingredients of a dough that is kneaded and baked to make the bakedproduct using methods well known in the art. See, for example, U.S. Pat.No. 4,567,046, EP-A-426,211, JP-A-60-78529, JP-A-62-111629, andJP-A-63-258528.

The term “effective amount” is defined herein as an amount of thealpha-amylase according to the invention that is sufficient forproviding a measurable effect on at least one property of interest ofthe dough and/or baked product. A suitable amount is in a range of10-20000 MU units/kg flour, in an embodiment 100-2000 MU/kg flour, in afurther embodiment 200-1000 MU/kg flour. A suitable amount includes 1ppm-2000 ppm of an enzyme having an activity in a range of about 10.000to 12.000. In an embodiment an effective amount is in a range of 10-200ppm of an enzyme having an activity in a range of about 10.000 to12.000, in another embodiment 20-80 ppm of an enzyme having an activityin a range of about 10.000 to 12.000. In an embodiment an effectiveamount is in a range of 10-200 ppm of an enzyme having an activity ofabout 10.000 MU/g. Herein and hereinafter MU stands for Maltotriose Unitas defined in the examples under the heading Maltotriose Assay (MUAssay).

The term “improved property” is defined herein as any property of adough and/or a product obtained from the dough, particularly a bakedproduct, which is improved by the action of the alpha-amylase enzymeaccording to the invention relative to a dough or product in which thealpha-amylase enzyme according to the invention is not incorporated. Theimproved property may include, but is not limited to, increased strengthof the dough, increased elasticity of the dough, increased stability ofthe dough, reduced stickiness of the dough, improved extensibility ofthe dough, improved machineability of the dough, increased volume of thebaked product, improved flavour of the baked product, improved crumbstructure of the baked product, improved crumb softness of the bakedproduct, reduced blistering of the baked product, improved crispiness,improved resilience both initial and in particular after storage,reduced hardness after storage and/or improved anti-staling of the bakedproduct.

The improved property may include faster dough development time of thedough and/or reduced dough stickiness of the dough.

The improved property may include improved foldability of the bakedproduct, such as improved foldability of a tortilla, a pancake, a flatbread, a pizza crust, a roti and/or a slice of bread.

The improved property may include improved flexibility of the bakedproduct including improved flexibility of a tortilla, a pancake, a flatbread, a pizza crust, a roti and/or a slice of bread.

The improved property may include improved stackability of flat bakedproducts including tortillas, pancakes, flat breads, pizza crusts, roti.

The improved property may include reduced stickiness of noodles and/orincreased flexibility of noodles.

The improved property may include reduced clumping of cooked noodlesand/or improved flavor of noodles even after a period of storage.

The improved property may include reduction of formation of hairlinecracks in a product in crackers as well as creating a leavening effectand improved flavor development.

The improved property may include improved mouth feel and /or improvedsoftness on squeeze,

The improved property may include reduced damage during transport,including reduced breaking during transport.

The improved property may include reduced hardness after storage ofgluten-free bread.

The improved property may include improved resilience of gluten-freebread. The improved property may include improved resilience bothinitial and in particular after storage of gluten-free bread.

The improved property may include reduced hardness after storage of ryebread.

The improved property may include reduced loss of resilience overstorage of rye bread,

The improved property may include reduced loss of resilience overstorage of a baked product comprising at least 5 wt % sugar, in anaspect comprising at least 8 wt % sugar, in an aspect comprising atleast 12 wt % sugar, in an aspect comprising at least 15 wt % sugarbased on flour. In an aspect comprising at least 18 wt % sugar, in anaspect comprising at least 20 wt % sugar, in an aspect comprising atleast 25 wt % sugar, in an aspect comprising at least 30 wt % sugarbased on flour. So for example 5% means 50 grams sugar per 1000 gram offlour used in the recipe.

The improved property may include reduced hardness after storage of abaked product comprising at least 5 wt % sugar, in an aspect comprisingat least 8 wt % sugar, in an aspect comprising at least 12 wt % sugar,in an aspect comprising at least 15 wt % sugar based on flour. In anaspect comprising at least 18 wt % sugar, in an aspect comprising aspectat least 20 wt % sugar, in an aspect comprising at least 25 wt % sugar,in an aspect comprising at least 30 wt % sugar based on flour. So forexample 5% means 50 grams sugar per 1000 gram of flour used in therecipe.

Improved mouth feel includes sense of softness on an initial bite orafter chewing, preferably without a sticky feeling in the mouth and/orwithout the baked product sticking to the teeth. Improved mouth feelincludes the baked product feeling less dry in the mouth on an initialbite or after chewing. Improved mouth feel includes the baked productfeeling less dry in the mouth on an initial bite or after chewing afterit has been kept outside its packaging or container. The improvedproperty may include that after a slice of bread was taken from itspackaging or container and exposed to ambient conditions for 5 minutes,in an aspect for 10 minutes, in an aspect for 20 minutes it has improvedmouthfeel.

The improved property may include that after a the cookie was taken fromits packaging or container and exposed to ambient conditions for 10minutes, in an aspect for 20 minutes, in an aspect for 30 minutes, in anaspect an hour it has improved mouthfeel. In an aspect ambientconditions herein and herein after include a temperature of 20 degreesC. and a moisture level of 40% humidity.

Reduced breaking during transport includes the baked product, includingwithout limitation cookies, bread such as gluten free bread, does notbreak in additional pieces as a consequence of transport.

Improved softness on squeeze includes the tactile experience that if abun is held between the fingers and the thumb of a hand and the thumband fingers are moved towards each other it takes less force.

Improved foldability of a baked product may be determined as follows.

The baked product is laid on a flat surface. The baked product is foldedby picking up one edge of the product and placing it on the oppositeedge of the product. This way a folded baked product is obtained havinga bend curve in an area located at or close to the center. The surfaceof the outside of the bend of folded baked product is visuallyinspected. The foldability is improved if fewer cracks are observed ator close to the bend. This may be a particularly useful property if thebaked product is a tortilla and/or a slice of bread.

Improved stackability may be determined as follows.

-   10 baked products are stacked on top of each other and sealed in a    polymer package, such as polyethylene foil. This yields a pack of    baked products. 10 packs of baked product are stacked on top of each    other and kept under ambient conditions for 3 days, in an aspect for    5 days in an aspect for 1 week, in an aspect for 2 weeks. Ambient    conditions are conditions as defined herein. After this period the    bottom pack of baked products is opened, the baked products are    separated from each other and the surfaces of the products are    visually inspected. The stackability is improved if less surface    damage is observed. Surface damage may be caused e.g. by rupture of    the surface during separation of two baked products that were    stacked on top of each other. This may be a particularly useful    property if the baked product is a tortilla.

Faster dough development time may be determined as follows

Dough development time is the time the dough need to reach maximumconsistency, maximum viscosity before gluten strands begin to breakdown. It may be determined by measuring peak time, using a Farinograph®from Brabender®, Germany. If a faronigraph is used to determine doughdevelopment time, dough development time is the time between the momentwater is added and the moment the curve reaches its highest point. Peaktime is preferably expressed in minutes.

Reduced dough stickiness may be determined as follows.

Dough stickiness is preferably determined on two separate batches of atleast 8 dough pieces, with the Texture Analyser TAXT2i (Stable MicroSystems Ltd., Surrey, UK) equipped with a 5 kg load cell in the measureforce in compression mode with a cylindrical probe (25 mm diameter).Using pre- and post-test speeds of 2.0 mm/s, while the test speed is 1.0mm/s. Dough pieces are centered and compressed 50% and the probe is heldfor 10 s at maximum compression. A negative peak value indicates doughstickiness. A less negative peak value indicates reduced doughstickiness.

Increased flexibility may be determined as follows.

The baked product is laid on a flat surface. The baked product is rolledto a shape similar to a pipe, this way a rolled baked product isobtained. The flexiblity is improved if the rolled baked product remainsits rolled up shape and does not roll open. This may be a particularlyuseful property if the baked product is a tortilla or a pancake.

The improved property may be determined by comparison of a dough and/ora baked product prepared with and without addition of the (isolated)polypeptide of the present invention in accordance with the methods ofpresent invention which are described below in the Examples.Organoleptic qualities may be evaluated using procedures wellestablished in the baking industry, and may include, for example, theuse of a panel of trained taste-testers.

The term “increased strength of the dough” is defined herein as theproperty of a dough that has generally more elastic properties and/orrequires more work input to mould and shape.

The term “increased elasticity of the dough” is defined herein as theproperty of a dough which has a higher tendency to regain its originalshape after being subjected to a certain physical strain.

The term “increased stability of the dough” is defined herein as theproperty of a dough that is less susceptible to forming faults as aconsequence of mechanical abuse thus better maintaining its shape andvolume and is evaluated by the ratio of height:width of a cross sectionof a loaf after normal and/or extended proof.

The term “reduced stickiness of the dough” is defined herein as theproperty of a dough that has less tendency to adhere to surfaces, e.g.,in the dough production machinery, and is either evaluated empiricallyby the skilled test baker or measured by the use of a texture analyser(e.g. a TAXT Plus) as known in the art.

The term “improved extensibility of the dough” is defined herein as theproperty of a dough that can be subjected to increased strain orstretching without rupture.

The term “improved machineability of the dough” is defined herein as theproperty of a dough that is generally less sticky and/or more firmand/or more elastic. Consequently there is less fouling of plantequipment and a reduced need for cleaning.

The term “increased volume of the baked product” is preferably measuredas the volume of a given loaf of bread determined by an automated breadvolume analyser (eg. BVM-3, TexVol Instruments AB, Viken, Sweden), usingultrasound or laser detection as known in the art. In case the volume isincreased, the property is improved. Alternatively the height of thebaked product after baking in the same size tin is an indication of thebaked product volume. In case the height of the baked product hasincreased, the volume of the baked product has increased.

The term “reduced blistering of the baked product” is defined herein asa visually determined reduction of blistering on the crust of the bakedbread.

The term “improved crumb structure of the baked product” is definedherein as the property of a baked product with finer cells and/orthinner cell walls in the crumb and/or more uniform/homogenousdistribution of cells in the crumb and is usually evaluated visually bythe baker or by digital image analysis as known in the art (eg. C-cell,Calibre Control International Ltd, Appleton, Warrington, UK).

The term “improved softness of the baked product” is the opposite of“hardness” and is defined herein as the property of a baked product thatis more easily compressed and is evaluated either empirically by theskilled test baker or measured by the use of a texture analyzer (e.g.TAXT Plus) as known in the art.

The term “improved flavor of the baked product” is evaluated by atrained test panel.

The term “improved anti-staling of the baked product” is defined hereinas the properties of a baked product that have a reduced rate ofdeterioration of quality parameters, e.g. reduced hardness after storageand/or decreased loss of resilience after storage.

Anti-staling properties may be demonstrated by a reduced hardness afterstorage of the baked product. The alpha-amylase according to theinvention may result in reduced hardness, e.g. in a baked product thatis more easily compressed. The hardness of the baked product may beevaluated either empirically by the skilled test baker or measured bythe use of a texture analyzer (e.g. TAXT Plus) as known in the art. Thehardness measured within 24 hours after baking is called initialhardness. The hardness measured 24 hours or more after baking is calledhardness after storage, and is also a measure for determining shelflife. In case the initial hardness has reduced, it has improved. In casethe hardness after storage has reduced, it has improved. Preferablyhardness is measured as described in example 9 herein.

Resilience of the baked product is preferably measured by the use of atexture analyzer (e.g. TAXTPlus) as known in the art.

The resilience measured within 24 hours after baking is called initialresilience. The resilience measured 24 hours or more after baking iscalled resilience after storage, and is also a measure for determiningshelf life. Freshly baked product typically gives crumb of high initialresilience but resilience is lost over shelf-life. Improved anti-stalingproperties may be demonstrated by a reduced loss of resilience overstorage. Preferably resilience is measured as described in example 9herein.

The term “improved crispiness” is defined herein as the property of abaked product to give a crispier sensation than a reference product asknown in the art, as well as to maintain this crispier perception for alonger time than a reference product. This property can be quantified bymeasuring a force versus distance curve at a fixed speed in acompression experiment using e.g. a texture analyzer TA-XT Plus (StableMicro Systems Ltd, Surrey, UK), and obtaining physical parameters fromthis compression curve, viz. (i) force of the first peak, (ii) distanceof the first peak, (iii) the initial slope, (iv) the force of thehighest peak, (v) the area under the graph and (vi) the amount offracture events (force drops larger than a certain preset value).Indications of improved crispness are a higher force of the first peak,a shorter distance of the first peak, a higher initial slope, a higherforce of the highest peak, higher area under the graph and a largernumber of fracture events. A crispier product should score statisticallysignificantly better on at least two of these parameters as compared toa reference product. In the art, “crispiness” is also referred to ascrispness, crunchiness or crustiness, meaning a material with a crispy,crunchy or crusty fracture behaviour.

The present invention may provide a dough having at least one of theimproved properties selected from the group consisting of increasedstrength, increased elasticity, increased stability, reduced stickiness,and/or improved extensibility of the dough.

The invention also may provide a baked product having increased loafvolume. The invention may provide as well a baked product having atleast one improved property selected from the group consisting ofincreased volume, improved flavour, improved crumb structure, improvedcrumb softness, improved crispiness, reduced blistering and/or improvedanti-staling.

The alpha-amylase according to the invention may be used for retardingstaling of a baked product such as bread and cake. Retarding of stalingmay be indicated by a reduced hardness, in particular a reduced hardnessafter storage compared to a baked product, including bread and cake,that is produced without the alpha-amylase according to the inventionaccording to the invention.

The alpha-amylase according to the invention has an intermediatethermostability compared with other alpha-amylases used in the industry.The alpha-amylase of the invention has higher temperature stability thanfungal alpha-amylase or alpha amylase from cereal flour. On the otherhand, it has a lower thermostability at high temperature, in particulara lower thermostability at the inactivation temperature of thealpha-amylase during baking, than other amylases used in the industry,such as bacterial alpha-amylase.

The alpha-amylase according to the invention has a lower thermostabilityat a high temperature, in an embodiment at a temperature above 70° C.,preferably at a temperature above 75° C., preferably at a temperatureabove 78° C., preferably at a temperature above 80° C., preferably at atemperature above 82° C., preferably at a temperature above 85° C.,compared to known alpha-amylases as measured using a method as describedin example 8 herein. Preferably thermostability is evaluated as follows:25 MU/ml purified enzyme solution in a buffer containing 50 mM sodiumacetate, pH 5.0, 1 mM CaCl₂ and 1 g/L BSA are pre-incubated in anEppendorf tube for 30 minutes at various temperatures (40° C. to 86°C.). The residual enzyme activity is determined using the MU assaydescribed herein in the examples under “Determination of enzymeactivity”, “2) Maltotriose assay (MU assay)”.

The alpha-amylase enzyme according to the invention is preferably activeduring baking and is preferably inactivated before end of baking.

Benefits of the alpha-amylase according to the invention having anintermediate thermostability and/or a lower thermostability at a hightemperature, may include, without limitation, one or more of thefollowing.

An enzyme having lower thermostability at a high temperature may resultin an increased level of denaturation of the enzyme during the bakingprocess. This may result in a more complete inactivation of the enzymeactivity and thus impart greater control of enzyme function in thebaking process.

It has been observed that small and large baked products have differentheat transfer rates, different bake times and consequently differentthermal treatments. The alpha-amylase according to the invention may bebeneficial for baked products undergoing less thermal treatment as aconsequence of reduced baking time and/or temperature.

It has been observed that bread baked at a higher altitude such aslocations above 2000 m (e.g. Mexico City 2240 m altitude) may sufferfrom difficulties in achieving crumb temperatures sufficient toinactivate thermostable enzymes. Without being bound to theory, it isthought that this is because the water boils at a lower temperature dueto the lower atmospheric pressure, and this dictates the maximumtemperature reached in the centre of a baked product. A lower maximumtemperature in the centre of the baked product may make it moredifficult to (fully) inactivate the enzyme. An alpha-amylase havinglower thermostability at high temperature might confer advantage in suchlocations, such as Mexico City, for example.

Industrial bakeries are under increasing pressure to reduce baking timesand oven temperatures—often to below 20 minutes, both for cost benefitand for sustainability reasons. A more heat labile enzyme may be bettersuited to a shorter baking time in that the enzyme is more effectivelydenatured at the end of the baking process. Parbaked bread receives ashorter baking time—e.g. typically 20% shorter baking process and/or 10°C. lower oven temperature than full baked equivalents, and may thereforealso be expected to benefit from an enzyme having lower thermostabilityat high temperatures.

The alpha-amylase enzyme of the present invention and/or additionalenzymes to be used in the methods of the present invention may be in anyform suitable for the use in question, e.g. in the form of a dry powder,agglomerated powder or granulate, in particular a non-dusting granulate,liquid, in particular a stabilized liquid, or protected enzyme suchdescribed in WO01/11974 and WO02/26044. A liquid form includes withoutlimitation an emulsion, a suspension and a solution. Granulates andagglomerated powders may be prepared by conventional methods, e.g. byspraying the alpha-amylase enzyme according to the invention onto acarrier in a fluid-bed granulator. The carrier may consist ofparticulate cores having a suitable particle size. The carrier may besoluble or insoluble, suitable carriers include a salt (such as NaCl orsodium sulphate), sugar alcohol (such as sorbitol), starch, rice flour,wheat flour, corn grits, maltodextrins or soy.

Such granulate or agglomerated powder, comprising the polypeptide of thepresent invention, may be referred to as a baking additive. The bakingadditive preferably has a narrow particle size distribution with morethan 95% (by weight) of the particles in the range from 25 to 500 μm.

The amylolytic enzyme according to the invention and/or additionalenzymes may be contained in slow-release formulations. Methods forpreparing slow-release formulations are well known in the art. Addingnutritionally acceptable stabilizers such as sugar, sugar alcohol, oranother polyol, and/or lactic acid or another organic acid according toestablished methods may for instance, stabilize liquid enzymepreparations.

Preferably the enzyme according to the invention is provided in a dryform, to allow easy handling of the product. Irrespective of theformulation of the enzyme, the formulation may comprise one or moreadditives. Examples of suitable additives include oxidants (includingascorbic acid, bromate and Azodicarbonamide (ADA)), reducing agents(including L-cysteine), emulsifiers (including mono/di glycerides,monoglycerides such as glycerol monostearate (GMS), sodium stearoyllactylate (SSL), calcium stearoyl lactylate (CSL), polyglycerol estersof fatty acids (PGE) and diacetyl tartaric acid esters of mono- anddiglycerides (DATEM), gums (including guargum and xanthangum), flavours,acids (including citric acid, propionic acid), starch, modified starch,gluten, humectants (including glycerol) and preservatives.

The alpha-amylase enzyme according to the invention may also beincorporated in yeast comprising compositions such as disclosed inEP-A-0619947, EP-A-0659344 and WO02/49441.

For inclusion in a pre-mix of flour it is advantageous that the(isolated) polypeptide according to the invention is in the form of adry product, e.g., a non-dusting granulate, whereas for inclusiontogether with a liquid it is advantageously in a liquid form.

One or more additional enzymes may also be incorporated into the dough.Therefore the invention provides an enzyme composition comprising thealpha-amylase enzyme according to the invention and one or moreadditional enzymes. The enzyme composition may be a baking enzymecomposition. This enzyme composition may be used in dough products andbaked products obtained from such dough. For example it may used indough products further containing eggs and in baked products, such asbrioche and panettone, both regular and with a reduced amount of eggs.The additional enzyme may be of any origin, including mammalian andplant, and preferably of microbial (bacterial, yeast or fungal) originand may be obtained by techniques conventionally used in the art.

In an embodiment, the additional enzyme may be an amylase, including afurther alpha-amylase, such as an fungal alpha-amylase (which may beuseful for providing sugars fermentable by yeast and retarding staling),beta-amylase, a cyclodextrin glucanotransferase, a protease, apeptidase, in particular, an exopeptidase (which may be useful inflavour enhancement), transglutaminase, triacyl glycerol lipase (whichmay be useful for the modification of lipids present in the dough ordough constituents so as to soften the dough), galactolipase,phospholipase, cellulase, hemicellulase, in particular a pentosanasesuch as xylanase (which may be useful for the partial hydrolysis ofpentosans, more specifically arabinoxylan, which increases theextensibility of the dough), protease (which may be useful for glutenweakening in particular when using hard wheat flour), protein disulfideisomerase, e.g., a protein disulfide isomerase as disclosed in WO95/00636, glycosyltransferase, peroxidase (which may be useful forimproving the dough consistency), laccase, or oxidase, hexose oxidase,e.g., a glucose oxidase, aldose oxidase, pyranose oxidase, lipoxygenaseor L-amino acid oxidase (which may be useful in improving doughconsistency) or a protease.

The cellulase may be from A. niger or from Trichoderma reesei.

The amyloglucosidase, may be an amyloglucosidase from Aspergillus suchas from A. oryzae or A. niger, preferably from A. niger.

In an embodiment the additional enzyme is a lipolytic enzyme, includinga triacyl glycerol lipase, a phospholipase, a galactolipase and anenzyme having both galactolipase and phospholipase activity.

The triacyl glycerol lipase may be a fungal lipase, preferably fromRhizopus, Aspergillus, Candida, Penicillum, Thermomyces, or Rhizomucor.In an embodiment the triacyl glycerol lipase is from Rhyzopus, in afurther embodiment a triacyl glycerol lipase from Rhyzopus oryzae isused. Optionally a combination of two or more triacyl glycerol lipasesmay be used.

In a further embodiment the lipolytic enzyme is a phospholipase or anenzyme having both galactolipase and phospholipase activity. Suchlipases are known to be active on the endogenous lipids of wheat and onextraneous lipid sources, for example as provided by added shorteningfat or from lecithin. Preferentially the lipase cleaves polar lipids andhas phospholipase activity, galactolipase activity or a combination ofphospholipase and galactolipase activity to create lysophospholipids,such as lysophoshotidyl choline, and lysogalactolipids such asdigalactosylmonoglyceride. The specificity of the lipase can be shownthrough in vitro assay making use of appropriate substrate, for exampletriacylglycerol lipid, phosphotidylcholine and diglactosyldiglyceride,or preferably through analysis of the reactions products that aregenerated in the dough during mixing and fermentation.

Panamore®, Lipopan® F, Lipopan® 50 and Lipopan® S are commercialised tostandardised lipolytic activity, using a measurement of DLU forPanamore® from DSM and a measurement of LU for the Lipopan® family fromNovozymes. DLU is defined as the amount of enzyme needed to produce 1micromol/min of p-nitrophenol from p-nitrophenyl palmitate at pH 8.5 at37° C., while LU is defined as the amount of enzyme needed to produce 1micromol/min of butyric acid from tributyrin at pH 7 at 30° C. Lipasesare optimally used with the alpha-amylase of the invention at 2-850DLU/kg flour or at 50-23500 LU/kg flour.

In an embodiment of the enzyme composition according to the inventionthe additional enzyme is Panamore® as described in WO2009/106575.

In an embodiment of the enzyme composition of the invention theadditional enzyme is an enzyme as described in WO9826057.

In an aspect of the enzyme composition according to the invention theadditional enzyme is an enzyme as described in U.S. Pat. No. RE38,507.

In an aspect of the enzyme composition according to the invention theadditional enzyme is an enzyme as described in WO 9943794, in particularin EP1058724B1.

If one or more additional enzyme activities are to be added inaccordance with the methods of the present invention, these activitiesmay be added separately or together with the polypeptide according tothe invention, for example as the enzyme composition according to theinvention, which includes a bread-improving composition and/or adough-improving composition. The other enzyme activities may be any ofthe enzymes described above and may be dosed in accordance withestablished baking practices.

In an embodiment the enzyme composition according to the invention isprovided in a dry form, to allow easy addition to the dough, the doughingredients, but liquid forms are also possible. A liquid form includeswithout limitation an emulsion, a suspension and a solution.Irrespective of the formulation of the enzyme composition, any additiveor additives known to be useful in the art to improve and/or maintainthe enzyme's activity, the quality of the dough and/or the baked productmay be applied. Examples of suitable additives include oxidants(including ascorbic acid, bromate and Azodicarbonamide (ADA)), reducingagents (including L-cysteine), emulsifiers (including mono/diglycerides, monoglycerides such as glycerol monostearate (GMS), sodiumstearoyl lactylate (SSL), calcium stearoyl lactylate (CSL), polyglycerolesters of fatty acids (PGE) and diacetyl tartaric acid esters of mono-and diglycerides (DATEM), gums (including guargum and xanthangum),flavours, acids (including citric acid, propionic acid), starch,modified starch, gluten, humectants (including glycerol) andpreservatives.

The alpha-amylase according to the invention may be incorporated in apre-mix, e.g. in the form of a flour composition, for dough and/or bakedproducts made from dough, in which the pre-mix comprises a polypeptideof the present invention. The term “pre-mix” is defined herein to beunderstood in its conventional meaning, i.e. as a mix of baking agents,generally including flour, which may be used not only in industrialbread-baking plants/facilities, but also in retail bakeries. The pre-mixmay be prepared by mixing the alpha-amylase according to the inventionor the enzyme composition according to the invention with a suitablecarrier such as flour, starch or a salt. The pre-mix may containadditives as mentioned above.

In another aspect, the alpha-amylase enzyme according to the inventionmay be used in the production of cake and in the production of a batterfrom which a cake can be made.

In another aspect, the alpha-amylase enzyme according to the inventionmay be used to reduce hardness after storage of a baked productcontaining at least 10 wt % sugar based on flour. So for example 5%means 50 grams per 1000 gram of flour used in the recipe.

The alpha-amylase enzyme according to the invention may be used in thepreparation of a wide range of cakes, including shortened cakes, such asfor example pound cake and butter cake, and including foam cakes, suchas for example meringues, sponge cake, biscuit cake, roulade, genoiseand chiffon cake. Sponge cake is a type of soft cake based on wheatflour, sugar, baking powder and eggs (and optionally baking powder). Theonly fat present is from the egg yolk, which is sometimes addedseparately from the white. It is often used as a base for other types ofcakes and desserts. A pound cake is traditionally prepared of one poundeach of flour, butter, eggs, and sugar, optionally complemented withbaking powder. In chiffon cake the butter/margarine has been replaced byoil. Sugar and egg yolk content has been decreased compared to pound orsponge cake and egg white content has been increased.

A method to prepare a batter preferably comprises the steps of:

-   -   a. preparing the batter of the cake by adding at least:        -   i. sugar;        -   ii. flour;        -   iii. the alpha-amylase enzyme according to the invention;        -   iv. at least one egg; and        -   v. optionally a phospholipase.

A method to prepare a cake according to the invention further comprisesthe step of

-   -   b. baking the batter to yield a cake.

The person skilled in the art knows how to prepare a batter or a cakestarting from dough ingredients. Optionally one or more otheringredients can be present in the composition e.g. to allow reduction ofeggs and/or fat in the cake, such as hydrocolloids, yeast extract,calcium.

The above-mentioned industrial applications of the alpha-amylase enzymeaccording to the invention comprise only a few examples and this listingis not meant to be restrictive.

Other uses of the alpha-amylase according to the invention may include:

-   -   the production of glucose, fructose and maltose syrups;    -   production of starch hydrolysates such as maltodextrins;    -   production of modified starches;    -   modification of starch components in animal feed;    -   replacement of malt in brewing;    -   use in a Glue including wall paper paste;    -   use in plastic objects made using starch, including plastic bags        made from polymerized starch films; and/or    -   use in waist bread reprocessing.

EXAMPLES Determination of Enzyme Activity 1) AACC Method 22-02.01Measurement of Alpha-Amylase in Plant and Microbial Materials Using theCeralpha® Method

The alpha-amylase activity was quantified by measuring activity using aMegazyme CERALPHA alpha-amylase assay kit (Megazyme InternationalIreland Ltd., Co. Wicklow, Ireland) according to the manufacturer'sinstruction.

2) Maltotriose Assay (MU Assay)

One Maltotriose Unit (MU) is defined as the amount of enzyme thatliberates 1 μmole glucose per minute using maltotriose substrate underthe following assay conditions. Enzymatic activity was determined at 37°C. and pH 5.0 using maltotriose as substrate. Enzymatic hydrolysis ofmaltotriose results in quantitative release of glucose, which is ameasure for enzymatic activity. The final assay concentrations: 8 mg/mlmaltotriose, 0.007 to 0.02 MU/ml mature DSM-AM, 20 mM citrate buffer,0.2 mg/ml BSA, 2 mM NaCl. The reaction was stopped after 30 minutes(addition of 0.33 M NaOH in 1:10 ratio) and the released glucose wasconverted into gluconate-6-P in two steps during which NADH is formed,using a Glucose Hexokinase FS kit (Diagn. Syst). The resultingabsorbance increases at a wavelength of 340 nm was a measure for theamount of glucose released during the 30 minute incubation. Activity wascalculated using a glucose calibration line.

Example 1 Production of the Alpha-Amylase of the Invention Cloning andEnzyme Preparation

As described in further detail below the alpha-amylase gene was clonedand expressed in B. subtilis in the following way

Strains and Plasmids

Bacillus subtilis strain BS154 (CBS 363.94) (ΔaprE, ΔnprE, amyE-, spo-)is described in Quax and Broekhuizen 1994 Appl Microbiol Biotechnol. 41:425-431.

The E. coli/B. subtilis shuttle vector pBHA12 is described in(WO2008/000632). Alicyclobacillus pohliae NCIMB14276 was described byImperio et al (Int. J. Syst. Evol. Microbiol 58:221-225, 2008).

Bacillus stearothermophilus C599 (NCIMB11873) is described inWO91/04669.

Molecular Biology Techniques

Molecular biology techniques known to the skilled person were performed(see: Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3rdEd., CSHL Press, Cold Spring Harbor, N.Y., 2001). Polymerase chainreaction (PCR) was performed on a thermocycler with PhusionHigh-Fidelity DNA polymerase (Finnzymes OY, Aspoo, Finland) according tothe instructions of the manufacturer.

Amylase Activity

Alpha-amylase activity in the broth of B. subtilis cultures wasquantified as described above according to AACC Method 22-02.01.

Sequencing of Alicyclobacillus pohliae Genome

The genome of Alicyclobacillus pohliae NCIMB14276 was sequenced byBaseClear (Leiden, The Netherlands). The DNA was fragmented (shearing)and DNA adapters were ligated to both ends of the DNA fragments. Twosets of Illumina GAllx sequence reads were obtained. One set consistedof paired-end reads, spanning a distance of around 250 (+−125)nucleotides. The second set consisted of mate pair reads, spanning adistance of around 4200 nucleotides (+−2100). On all Illumina GAllxsequence reads a quality filtering was applied based on Phred qualityscores. In addition, low quality and ambiguous nucleotides were trimmedoff from the remaining reads. The filtered paired-end and mate pairreads were used for ‘De novo’ assembly in the CLC Genomics Workbenchversion 4.6.1 or 4.7 (CLC bio, Aarhus, Denmark). In this way, a set ofpre-assembled contigs (contiguous sequences) were obtained. The contigswere arranged further (scaffolding) using SSPACE described by Boetzer etal. (Bioinformatics 27:578-579, 2011). The sequence analysis revealedthat the Alicyclobacillus pohliae NCIMB14276 genome contains a geneencoding an alpha-amylase enzyme named DSM-AM herein with the nucleotidesequence as set out in SEQ ID NO: 1, see also FIG. 3.

The corresponding DSM-AM protein encoded by SEQ ID NO.1 has the aminoacid sequence as set out in SEQ ID NO: 2, see also FIG. 4.

The nucleotide sequence of the codon optimized DSM-AM gene is set out inSEQ ID NO: 3, see also FIG. 5.

Example 2 Expression of A. pohliae DSM-AM Gene in Bacillus subtilis

An amyQ terminator and a PmeI restriction site were introduced in thepBHA12 vector by digesting pBHA12 with SphI and HindIII and cloning thefollowing DNA sequence5′-GCATGCGTTTAAACAAAAACACCTCCAAGCTGAGTGCGGGTATCAGCTTGGAGGTGCGTTTATTTTTTCAGCCGTATGACAAGGTCGGCATCAGAAGCTT-3′ (the 5′SphI and 3′HindIIIrestriction sites are underlined).

The fragment was cloned into pBHA12 which resulted in vector pGBB09(FIG. 1).

The DSM-AM gene was synthesised by GeneArt (Germany) and at the 5′ endthe PacI restriction site was added and at its 3′end the PmeIrestriction site was added. The DSM-AM gene was cloned into the Pad andPmeI digested pGBB09 vector which resulted in vector pGBB09DSM-AM1 (FIG.2). This vector was transformed to B. subtilis strain BS154. Thesequence of the plasmid was confirmed by DNA sequencing. The B. subtilisstrain BS154 containing pGBB09DSM-AM1 was named DSM-AMB154-1.

Example 3 Expression of DSM-AM with B. subtilis in Shake Flasks

B. subtilis strains DSM-AMB154-1 and BS154 were grown in a shake flask.These shake flasks contained 20 ml 2×TY medium composed of 1.6% (w/v)Bacto tryptone, 1% (w/v) Yeast extract and 0.5% (w/v) NaCl. The cultureswere shaken vigorously at 37° C. and 250 rpm for 16 hours and 0.2 mlculture medium was used to inoculate 20 ml SMM medium. SMM pre-mediumcontains 1.25% (w/w) yeast extract, 0.05% (w/w) CaCl2, 0.075% (w/w)MgCl2.6H2O, 15 μg/l MnSO4.4H2O, 10 μg/l CoCl2.6H2O, 0.05% (w/w) citricacid, 0.025% (w/w) antifoam 86/013 (Basildon Chemicals, Abingdon, UK).To complete SMM medium, 20 ml of 5% (w/v) maltose and 20 ml of a 200 mMNa-phosphate buffer stock solution (pH 6.8), both prepared andsterilized separately, were added to 60 ml SMM pre-medium. Thesecultures were incubated for 48 hours at 37° C. and 250 rpm. Thesupernatants were harvested and analysed for enzyme productivity. Thealpha-amylase activity of strain DSM-AMB154-1 was measured according toAACC Method 22-02.01 as described in above. The supernatant ofDSM-AMB154-1 contained alpha-amylase activity whereas the parent strainBS154 did not.

Example 4 Enzyme Preparation

Bacillus strain DSM-AMB154-1 was cultivated under aerobic conditions ina suitable fermentation medium.

The enzyme was secreted into the medium. The ensuing fermentation brothwas filtered to remove bacterial cells, debris from these cells andother solids. The filtrate containing the enzyme, thus obtained, wasthen concentrated by ultrafiltration to yield a concentrate containingmature DSM-AM.

Example 5 Enzyme Purification

The purification was performed using of the following steps. Theconcentrated fermentation broth obtained in example 4 containing matureDSM-AM was mixed with 50 mM HEPES buffer, pH 7.5 containing 400 mg/ml(NH₄)₂SO4 (1:1 ratio). The solution was stirred overnight at 4° C., andfollowed by centrifugation at 3220 rcf, 4° C. for 10 minutes. The pelletwas resuspended in 25 mM Tris buffer, pH7.5, and filtrated through 0.45μm filter. The conductivity of the solution was adjusted to 2 ms/cm byaddition of MilliQ water, and followed by pH adjustment to pH=7.5. Thesolution was concentrated by Vivaspin 20 ml Concentrator (SartoriusStedim) 10.000 MWCO, at 3220 rcf, 4° C. for 15 min. The solution wasapplied to a Q-Sepharose column equilibrated with 25 mM HEPES buffer, pH7.5. The protein was collected in flow-through. Flow-through wasre-applied to a Q-sepharose column equilibrated with 25 mM HEPES buffer,pH 9.5. Protein was eluted with a 0-1 M NaCl gradient. The purifiedmature DSM-AM enzyme was desalted by a PD-10 desalting column (GEHealthcare) using 25 mM Tris buffer, pH7.5.

Protein Determination

The protein concentration of the purified mature DSM-AM enzyme asobtained in example 5 was determined by BCA™ protein assay kit (Pierce)according to the manufacturer's instruction with the followingcondition: the ratio of the sample to WR reagent was 1:12, and theabsorbance of the mixture was determined at wavelength of 540 nm.

Example 6 Enzyme Properties

Dependence of the enzyme activity of the mature DSM-AM as obtained inexample 5, on pH was tested by the MU assay described above using areaction mixture in which pH was adjusted to different values (pH 4.0,4.3, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5). Two measurements with two finalenzyme concentrations, 0.01 and 0.018 MU/ml were taken. The results weredescribed as a relative activity. The pH optimum for mature DSM-AM wasfound to be at pH 5.0. The activities measured at the other indicatedpH's are shown in the table below. The value corresponds to an averagevalue of both measurements at different enzyme concentrations.

TABLE 6.1 pH 4.0 4.3 5.0 5.5 6.0 6.5 7.0 7.5 Relative activity 68% 76%100% 87% 71% 40% 21% 8%

Dependence of the mature DSM-AM enzyme activity on temperature wasdetermined using the MU assay described above with the reactiontemperatures set to 40° C., 50° C., 60° C., 65° C., 70° C., 75° C., 80°C. and 90° C. Mature DSM-AM was most active at 60-75° C. and the enzymelost approximately 90% of its activity when the temperature was above90° C.

Example 7 Alpha-Amylase Activity

The alpha-amylase activity of the mature DSM-AM as obtained in example5, was quantified by measuring activity using a Megazyme CERALPHAalpha-amylase assay kit (Megazyme International Ireland Ltd., Co.Wicklow, Ireland) according to the manufacturer's instruction.

TABLE 7.1 Activity in Activity in Ceralpha Ceralpha Sample Activityassay @ 40° C. assay @ 60° C. Mature DSM-AM 1095 MU/ml 124 U/ml 191 U/mlControl malt Estimated 1.9 U/ml  2.8 U/ml flour  Estimated 36 U/ml  43U/ml

Example 8 Thermostability of Mature DSM-AM

To evaluate the thermostability of the mature DSM-AM, as obtained inexample 5, 25 MU/ml purified enzyme solution in a buffer containing 50mM sodium acetate, pH 5.0, 1 mM CaCl₂ and 1 g/L BSA were pre-incubatedin an Eppendorf tube for 30 minutes at various temperatures (40° C. to86° C.). The residual enzyme activity was determined using the MU assaydescribed above. For comparison, Novamyl® 10.000 was included. Theresults were expressed as a percentage of the activity of a sample thatwas pre-incubated for 30 minutes at 4° C.

In the temperature range from 40° C. to 75° C. comparable high-levelactivities (above 90%) were observed for mature DSM-AM and Novamyl®10.000 (data not shown). The residual enzyme activities from thetemperature range of 76 to 86° C. are listed in the Table 8.1 below.These data clearly demonstrated that mature DSM-AM and Novamyl® 10.000are comparably thermostable at temperatures up to about 80° C. However,at the temperatures of about 80° C. and higher the residual enzymaticactivity of mature DSM-AM is lower than that of Novamyl® 10.000.

TABLE 8.1 Enzyme activity Temperature [° C.] 4 76 78 80 82 84 86 MatureDSM-AM 100% 91% 85% 69% 38% 14%  1% Novamyl ® 10.000 100% 87% 82% 72%54% 31% 10% Novamyl ® 10.000 was obtained from Novozymes, Denmark.

To further evaluate the thermostability of the mature DSM-AM, asobtained in example 5, the residual activity of 7.5 MU/ml mature DSM-AMwas tested in glass tube in a buffer of 50 mM sodium acetate, pH 4.3, 1mM CaCl₂. After incubation at 80° C. for 15 minutes, the residualactivity was determined as described above. Mature DSM-AM showed aresidual activity of 3% whereas Novamyl® 10.000 showed 13% residualactivity at the same conditions. The results are listed in Table 8.2

TABLE 8.2 Residual activity Temperature [° C.] 4 80 Mature DSM-AM 100% 3% Novamyl ® 10.000 100% 13% Novamyl ® 10.000 was obtained fromNovozymes, Denmark.

Example 9 Baking Experiment

The baking performance of the mature DSM-AM was tested in Dutch tinbread. Two ingredient lists were used (see Tabel 9.1). Recipe A was usedfor the results in Table 9.2, recipe B was used for the results in Table9.3. The Control in table 9.2 and 9.3 refers to a loaf of bread preparedusing recipe A and B respectively, and not containing mature DSM-AM.

In the baking experiments a concentrate of mature DSM-AM, which may beproduced as described in example 4, was dosed at 550-605 MU/kg flour.

The ingredients listed in Table 9.1 were mixed in a Diosna SP-12 mixerfor 4 minutes at speed 400 turns/min and thereafter at speed 1560turns/min for 10 min, to a final dough temperature of 27° C. The doughwas divided in 8 pieces of 840 g, rounded and proofed for 45 minutes at28° C. and 90% relative humidity.

Afterwards the dough pieces were moulded, and placed in greased tins(DGP-01) and proofed for 75 minutes at 35° C. at relative humidity of88%.

The fully proofed dough pieces were placed in an Wachtel Piccolo ovenand baked in a first phase at 280° C. for 7 minutes with initial steamaddition. After that the temperature was raised to 265° C./270° C. for28 minutes in a second phase.

Thereafter the oven was unloaded, the breads were depanned and placed ona rack to cool for at least 1 hour at ambient temperature, which istypically between 20 and 25° C. After 1-2 hours cooling, the breads werewrapped in polyethylene plastic bags.

Thereafter the breads were assessed.

The breads were kept in the plastic bags in between the hardnessmeasurements.

TABLE 9.1 Recipe A Recipe B Ingredient (grams) (grams) Type Flour(Kolibri*) 2400 Flour (Ibis)* 600 Flour Edelweiss* 4500 Fresh yeast 108180 Koningsgist* Salt 81 81 Bread improver 22.5 Basic tin (DGP-06)**Bread improver 135 Rich Bread Improver*** Water 2470 2585 ⁺/−1% Calciumpropionate 1.8 *Kolibri, Edelweiss and Ibis flour were obtained fromMeneba, the Netherlands. Koningsgist was obtained from AB Mauri, theNetherlands **basic bread improver comprising 20 ppm ascorbic acid (fromDSM Nutritional Products, Switzerland), 5 ppm Bakezyme ® P500 (fungalalpha-amylase from DSM, The Netherlands), 15 ppm Bakezyme ® HSP6000(fungal hemicellulase from DSM, The Netherlands) and Kolibri flour asmixing material. ***Rich Bread Improver comprising Soy flour (from SojaAustria, Austria) 32.9 wt %, Whey powder (from Vreugdenhill, TheNetherlands), 18 wt % Palm oil (100% palmoil, from Remia, TheNetherlands), 6 wt %, Rapeseed oil (from Aldoc B.V., The Netherlands), 3wt %, SSL (from Cognis Deutschland GmbH&Co. KG, Germany), 10 wt %,Kolibri flour (from Meneba, The Netherlands) 30.1 wt %

Measurement of Hardness

The bread was sliced with a bread slicer set at 2.1 cm slice distance.

The hardness was measured using a using a Texture Analyser TA-XTPIusfrom Stable Micro Systems apparatus and applying the following settings.

Settings

-   -   Test mode=Compression    -   Pre-test speed=3 mm/s    -   Test speed=1 mm/s    -   Post test speed 5 mm/s    -   Distance=5 mm    -   Hold time=10 sec    -   Trigger force=5 g

The hardness listed is the Force measured; the max peak value recordedin gram. Resilience is the Force (F) after 10 sec holding time dividedby max peak force multiply by 100.

Resilience=(F2/F1)×100

After cooling down to room temperature the volumes of the loaves weredetermined by an automated bread volume analyser (BVM-3, TexVolInstruments). The loaf volume of the control bread is defined as 100%.

The Consistency, Body, Development, Extensibility, Elasticity,Stickiness, of the dough were evaluated by an experienced baker andjudged as good.

Volume, crumb structure and crumb colour of the bread were judged by anexperienced baker as good.

Satisfactory results were obtained, that indicated a good dough and agood bread.

TABLE 9.2 Average values of three tests with recipe A. Relative VolumeHardness Hardness (%) day 0 day 4 day 7 Control 100 529 653 Mature 101374 525 DSM-AM (550 MU/kg flour)

Day 0 is the day the bread was baked. Day 4 is the 4th day after thebread was baked. Day 7 is the 7th day after the bread was baked.

TABLE 9.3 Longer shelf life tests with Edelweiss (recipe B) RelativeVolume Hardness Hardness Hardness (%) day 0 week 1 week 2 week 3 Control100 429 592 672 Mature 101 263 426 516 DSM-AM (605 MU/kg flour)

Day 0 is the day the bread was baked. Week 1 is the 7^(th) day after thebread was baked. Week 2 is the 14^(th) day after the bread was baked.Week 3 is the 21^(st) day after the bread was baked.

From these results it can be seen that the hardness of the bread sliceswhen prepared using the mature DSM-AM is reduced after storage ascompared to the control bread slices which lack this enzyme.

Example 10 Baking Experiment

The baking performance of the mature DSM-AM was tested in open top tinbread containing higher levels of sugar. For comparison, Novamyl® 10.000was included. The sugar added was in the range of 12 to 20%. Theingredients used in the baking experiment are listed in Table 10.1. Theresults are shown in Table 10.2. The Control in table 10.2 refers to aloaf of bread prepared not containing mature DSM-AM or Novamyl® 10.000.

In the baking experiments a concentrate of mature DSM-AM, which may beproduced as described in example 4, was dosed at 550 MU/kg flour. Incomparison Novamyl® 10.000 was added at 50 mg/kg flour

The ingredients listed in Table 10.1 were mixed in a Diosna SP-12 mixer400 turns at a frequency of 25 Hz and thereafter 1800 turns at afrequency of 50 Hz, to a final dough temperature of 27° C. The dough wasdivided in 8 pieces of 840 g, rounded and proofed for 45 minutes at 28°C. and 90% relative humidity.

Afterwards the dough pieces were moulded, and placed in greased tins(DGP-01) and proofed for 75 minutes at 35° C. at relative humidity of88%.

The fully proofed dough pieces were placed in a Wachtel Piccolo oven andbaked in a first phase at 200/230° C. for 15 minutes with initial steamaddition. After that the temperature was decreased to 160/180° C. for 20minutes in a second phase.

Thereafter the oven was unloaded, the breads were depanned and placed ona rack to cool for at least 1 hour at ambient temperature, which istypically between 20 and 25° C. After 1-2 hours cooling, the breads werewrapped in polyethylene plastic bags.

Thereafter the breads were assessed.

The breads were kept in the plastic bags in between the hardnessmeasurements.

TABLE 10.1 Ingredient Recipe (grams) Type Flour (BG100*) 4500 Freshyeast 108 Koningsgist* Salt 81 Bread improver 22.5 Basic tin (DGP-06)**Sugar 540-720-900 Water 2430 ⁺/−1% *BG100 flour was obtained fromPaniflour, Belgium. Koningsgist was obtained from AB Mauri, theNetherlands **basic bread improver comprising 20 ppm ascorbic acid (fromDSM Nutritional Products, Switzerland), 5 ppm Bakezyme ® P500 (fungalalpha-amylase from DSM, The Netherlands), 15 ppm Bakezyme ® HSP6000(fungal hemicellulase from DSM, The Netherlands) and Kolibri flour asmixing material.

Measurement of Hardness

The bread was sliced with a bread slicer set at 2.1 cm slice distance.

The hardness was measured using a using a Texture Analyser TA-XTPIusfrom Stable Micro Systems apparatus and applying the following settings.

Settings

-   -   Test mode=Compression    -   Pre-test speed=3 mm/s    -   Test speed=1 mm/s    -   Post test speed 5 mm/s    -   Distance=5 mm    -   Hold time=10 sec    -   Trigger force=5 g

The hardness listed is the Force measured; the max peak value recordedin gram. Resilience is the Force (F) after 10 sec holding time dividedby max peak force multiply by 100.

Resilience=(F2/F1)×100

After cooling down to room temperature the volumes of the loaves weredetermined by an automated bread volume analyser (BVM-3, TexVolInstruments). The loaf volume of the control bread is defined as 100%.

The Consistency, Body, Development, Extensibility, Elasticity,Stickiness, of the dough were evaluated by an experienced baker andjudged as good.

Volume, crumb structure and crumb colour of the bread were judged by anexperienced baker as good.

Satisfactory results were obtained, that indicated a good dough and agood bread.

TABLE 10.2 Shelflife test Mature DSM-AM Control (550 MU/kg flour)Hardness day 4 480 252 12 wt % sugar Hardness day 7 603 316 12 wt %sugar Hardness day 4 530 341 16 wt % sugar Hardness day 7 637 400 16 wt% sugar Hardness day 4 977 553 20 wt % sugar Hardness day 7 1209 726 20wt % sugar

Day 0 is the day the bread was baked. Day 4 is the 4th day after thebread was baked. Day 7 is the 7th day after the bread was baked.

From these results it can be seen that the hardness of the bread sliceswhen prepared using the mature DSM-AM is reduced after storage ascompared to the control bread slices which lack this enzyme.

Example 11 Baking Experiment

The baking performance of the mature DSM-AM was tested in Dutch tinbread. Two ingredient lists were used (see Table 11.1). Recipe A wasused for the results in Table 11.2, recipe B was used for the results inTable 11.3. The Control in table 11.2 and 11.3 refers to a loaf of breadprepared using recipe A and B respectively, and not containing matureDSM-AM.

In the baking experiments a concentrate of mature DSM-AM, which may beproduced as described in example 4, was dosed at 550-605 MU/kg flour.

The ingredients listed in Table 11.1 were mixed in a Diosna SP-12 mixer400 turns at a frequency of 25 Hz and thereafter 1800 turns at afrequency of 50 Hz, to a final dough temperature of 27° C. The dough wasdivided in 8 pieces of 840 g, rounded and proofed for 45 minutes at 28°C. and 90% relative humidity.

Afterwards the dough pieces were moulded, and placed in greased tins(DGP-01) and proofed for 75 minutes at 35° C. at relative humidity of88%.

The fully proofed dough pieces were placed in an Wachtel Piccolo ovenand baked in a first phase at 280° C. for 7 minutes with initial steamaddition. After that the temperature was raised to 265° C./270° C. for28 minutes in a second phase.

Thereafter the oven was unloaded, the breads were depanned and placed ona rack to cool for at least 1 hour at ambient temperature, which istypically between 20 and 25° C. After 1-2 hours cooling, the breads werewrapped in polyethylene plastic bags.

Thereafter the breads were assessed.

The breads were kept in the plastic bags in between the hardnessmeasurements.

TABLE 11.1 Recipe A Recipe B Ingredient (grams) (grams) Type Flour(Kolibri*) 3600 Flour (Ibis)* 900 Flour Edelweiss* 4500 Fresh yeast 108180 Koningsgist* Salt 81 81 Bread improver 22.5 Basic tin (DGP-06)**Bread improver 135 Rich Bread Improver*** Water 2470 2585 ⁺/−1% Calciumpropionate 1.8 *Kolibri, Edelweiss and Ibis flour were obtained fromMeneba, the Netherlands. Koningsgist was obtained from AB Mauri, theNetherlands **basic bread improver comprising 20 ppm ascorbic acid (fromDSM Nutritional Products, Switzerland), 5 ppm Bakezyme ® P500 (fungalalpha-amylase from DSM, The Netherlands), 15 ppm Bakezyme ® HSP6000(fungal hemicellulase from DSM, The Netherlands) and Kolibri flour asmixing material. ***Rich Bread Improver comprising Soy flour (from SojaAustria, Austria) 32.9 wt %, Whey powder (from Vreugdenhill, TheNetherlands), 18 wt % Palm oil (100% palmoil, from Remia, TheNetherlands), 6 wt %, Rapeseed oil (from Aldoc B.V., The Netherlands), 3wt %, SSL (from Cognis Deutschland GmbH&Co. KG, Germany), 10 wt %,Kolibri flour (from Meneba, The Netherlands) 30.1 wt %

Measurement of Hardness

The bread was sliced with a bread slicer set at 2.1 cm slice distance.

The hardness was measured using a using a Texture Analyser TA-XTPIusfrom Stable Micro Systems apparatus and applying the following settings.

Settings

-   -   Test mode=Compression    -   Pre-test speed=3 mm/s    -   Test speed=1 mm/s    -   Post test speed 5 mm/s    -   Distance=5 mm    -   Hold time=10 sec    -   Trigger force=5 g

The hardness listed is the Force measured; the max peak value recordedin gram. Resilience is the Force (F) after 10 sec holding time dividedby max peak force multiply by 100.

Resilience=(F2/F1)×100

After cooling down to room temperature the volumes of the loaves weredetermined by an automated bread volume analyser (BVM-3, TexVolInstruments). The loaf volume of the control bread is defined as 100%.

The Consistency, Body, Development, Extensibility, Elasticity,Stickiness, of the dough were evaluated by an experienced baker andjudged as good.

Volume, crumb structure and crumb colour of the bread were judged by anexperienced baker as good.

Satisfactory results were obtained, that indicated a good dough and agood bread.

TABLE 11.2 Average values of three tests with recipe A. Relative VolumeHardness Hardness (%) day 0 day 4 day 7 Control 100 529 653 Mature 101374 525 DSM-AM (550 MU/kg flour)

Day 0 is the day the bread was baked. Day 4 is the 4th day after thebread was baked. Day 7 is the 7th day after the bread was baked.

TABLE 11.3 Longer shelf life tests with Edelweiss (recipe B) RelativeVolume Hardness Hardness Hardness (%) day 0 week 1 week 2 week 3 Control100 429 592 672 Mature 101 263 426 516 DSM-AM (605 MU/kg flour)

Day 0 is the day the bread was baked. Week 1 is the 7^(th) day after thebread was baked. Week 2 is the 14^(th) day after the bread was baked.Week 3 is the 21^(st) day after the bread was baked.

From these results it can be seen that the hardness of the bread sliceswhen prepared using the mature DSM-AM is reduced after storage ascompared to the control bread slices which lack this enzyme.

1. A polynucleotide encoding for a polypeptide having alpha-amylaseactivity comprising: (a) a polynucleotide sequence encoding apolypeptide having an amino acid sequence as set out in amino acids 34to 719 of SEQ ID NO: 2; or (b) a polynucleotide sequence encoding apolypeptide having at least 99.5% identity to a polypeptide having anamino acid sequence as set out in amino acids 34 to 719 of SEQ ID NO: 2;or (c) a polynucleotide sequence as set out in nucleotides 100 to 2157of SEQ ID NO: 1 or SEQ ID NO: 3; or (d) a polynucleotide sequence as setout in SEQ ID NO: 1 or SEQ ID NO:
 3. 2. The polynucleotide according toclaim 1, wherein the polynucleotide is produced by Alicyclobacilluspohliae NCIMB14276.
 3. A vector comprising the polynucleotide sequenceaccording to claim
 1. 4. The vector according to claim 3 which is anexpression vector wherein the polynucleotide sequence is operably linkedwith at least one regulatory sequence allowing for expression of thepolynucleotide sequence in a suitable host cell.
 5. The vector accordingto claim 4, wherein the suitable host cell is an Aspergillus, Bacillus,Chrysosporium, Escherichia, Kluyveromyces, Penicillium, Pseudomonas,Saccharomyces, Streptomyces or Talaromyces species, preferably aBacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis,Escherichia coli, Aspergillus Niger or Aspergillus oryzae species.
 6. Arecombinant host cell comprising the polynucleotide according toclaim
 1. 7. The recombinant host cell according to claim 6 capable ofexpressing or over-expressing said polynucleotide or a vector comprisingsaid polynucleotide.
 8. A method for manufacturing the polynucleotideaccording to claim 1 comprising culturing a host cell transformed withsaid polynucleotide or a vector thereof and isolating saidpolynucleotide or said vector from said host cell.
 9. An alpha-amylasepolypeptide comprising: (a) an amino acid sequence as set out in aminoacids 34 to 719 of SEQ ID NO: 2; or (b) an amino acid sequence having atleast 99.5% identity to an amino acid sequence as set out in amino acids34 to 719 of SEQ ID NO: 2; or (c) an amino acid sequence encoded by apolynucleotide as set out in nucleotides 100 to 2157 of SEQ ID NO: 1 orSEQ ID NO: 3; or (d) an amino acid sequence having at least 70% identityto an amino acid sequence as set out in amino acids 34 to 719 of SEQ IDNO: 2 and having at least one of Asp at position 184, Ala at position297, Thr at position 368 and Asn at position 489, said positions beingdefined with reference to SEQ ID NO: 2; or (e) an amino acid sequencehaving at least 70% identity to an amino acid sequence as set out inamino acids 34 to 719 of SEQ ID NO: 2 and having at least one of Asp atposition 184, Ala at position 297, Thr at position 368 and Asn atposition 489, said positions being defined with reference to SEQ ID NO:2 and said amino acid sequence characterized in that when used toprepare a baked product having a least 5 wt % sugar based on flour, saidbaked product has reduced hardness after storage in comparison with abaked product prepared without use of said amino acid sequence.
 10. Thepolypeptide according to claim 9 obtainable by expressing thepolynucleotide a vector comprising said polynucleotide in an appropriatehost cell.
 11. A method for manufacturing the polypeptide according toclaim 9 comprising cultivating a recombinant host cell under conditionwhich allow for expression of the polynucleotide or a vector comprisingsaid polynucleotide and, optionally, recovering an encoded polypeptidefrom the cell or culture medium.
 12. A polypeptide according to claim 9capable of being used in food manufacturing.
 13. A polypeptide accordingto claim 12 capable of being used in manufacture of a baked product,optionally a bread or a cake.
 14. Enzyme composition comprising thepolypeptide according to claim 9 and one or more components selectedfrom the group consisting of milk powder, gluten, granulated fat, anadditional enzyme, an amino acid, a salt, an oxidant such as ascorbicacid, bromate and azodicarbonamide, a reducing agent such as L-cysteine,an emulsifier such as mono-glycerides, di-glycerides, glycerolmonostearate, sodium stearoyl lactylate, calcium stearoyl lactylate,polyglycerol esters of fatty acids and diacetyl tartaric acid esters ofmono- and diglycerides, gums such as guargum and xanthangum, flavours,acids such as citric acid and propionic acid, starch, modified starch,gluten, humectants such as glycerol, and preservatives.
 15. Enzymecomposition according to claim 14, wherein the additional enzyme is alipolytic enzyme, optionally a phospholipase.
 16. Method to prepare adough comprising the step of combining the polypeptide according toclaim 9 and at least one dough ingredient.
 17. A dough comprising thepolypeptide according to claim
 9. 18. Method to prepare a baked productcomprising baking the dough according to claim
 17. 19. Baked productobtainable by the method according to claim
 18. 20. A method to producea polypeptide having at least 60% identity to (a) an amino acid sequenceas set out in amino acids 34 to 719 of SEQ ID NO: 2 or an amino acidsequence having at least 99.5% identity to amino acids 34 to 719 of theamino acid sequence of SEQ ID NO: 2; or (b) an amino acid sequenceencoded by the polynucleotide according to claim 1, comprising usingAlicyclobacillus pohliae NCIMB14276.
 21. The polypeptide according toclaim 9 capable of being used to reduce hardness after storage of abaked product comprising at least 5 wt % sugar based on flour.